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Biotechnology
Biotechnology
Second Edition
Wallace Woon-Fong Leung

Elsevier
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Contents
In God, I trust xvii

Preface to Second Edition (2019) xix
Preface to First Edition (2007) xxiii
1 Introduction 1
1.1 Introduction 1
1.1.1 Common Host Cells for Secreting Recombinant
Protein 6
1.1.2 Platform for Protein Expression 8
1.1.3 Extracellular Protein 9
1.1.4 Intracellular Protein—Liquid 9
1.1.5 Intracellular Protein—Inclusion Body 10
1.2 Centrifugal Separation and Filtration 12
1.2.1 Sedimenting Centrifuge 14
1.2.2 Filtering Centrifuges 15
1.3 Pros and Cons of Filtration Versus Centrifugation 16
1.4 Generic Flow Sheet for Biopharmaceutical Process 18
1.5 Other Centrifugal Separations 19
1.6 Inputs and Outputs of Centrifuge 19
1.7 Separation Metrics 20
1.7.1 Protein Yield 20
1.7.2 Centrate Suspended Solids 20
1.7.3 Throughput Rate 21
1.7.4 Cell Viability 21
1.8 Text Organization 22
1.9 Summary 23
References 23
Problems 25
v
vi CONTENTS
2 Principles of Centrifugal Sedimentation 27

2.1 Introduction 27
2.2 Nonintuitive Phenomena 27
2.2.1 Pressure Gradient in a Fluid Under Centrifugal
Acceleration 27
2.2.2 Combined Centrifugal and Gravitational
Accelerations 28
2.2.3 Coriolis Effect 32
2.3 Intuitive Phenomena 35
2.3.1 Centrifugal Acceleration 35
2.3.2 Fluid in a Centrifuge Bowl Not at Solid-Body
Rotation 38
2.3.3 Regimes of Sedimentation 40
2.3.4 Stokes’ Law 42
2.3.5 Settling With Concentrated Solids 43
2.4 Process Functions 44
2.5 Summary 46
References 46
Problems 47
3 Batch and Semibatch Centrifuges 49

3.1 Spintube 49
3.2 Centrifugal Filter 53
3.3 Ultracentrifuges 54
3.3.1 Analytical Ultracentrifuge 55
3.3.2 Preparative Ultracentrifuge 56
3.3.3 Centrifugal Elutriation 58
3.4 Tubular Centrifuge 60
3.4.1 General Tubular Bowl Geometry 60
3.4.2 Ribs and Solids Scraper 63
3.4.3 Automatic Plunger Cake Discharge 67
3.5 Summary 69
References 70
Problems 70
4 Disk Centrifuge 73
4.1 Lamella/Inclined Plate Settler 73
4.1.1 Inclined Plate Settler Principle 73
4.1.2 Complications in Inclined Plate Settler 74
CONTENTS vii
4.2 Disk-Stack Centrifuge 75

4.2.1 General Disk Geometry 75
4.2.2 Disk Angle 77
4.2.3 Disk Spacing 77
4.2.4 Process Functions of Disk Centrifuge 79
4.2.5 Feed Solids 80
4.2.6 Manual Disk Centrifuge 80
4.2.7 Intermittent Discharge 82
4.2.8 Chamber Bowl 87
4.2.9 Continuous Concentrate Discharge 87
4.2.10 Liquid Discharge 95
4.2.11 Solution to Adverse Heating Effect 100
4.3 Feed Inlet and Accelerator 100
4.3.1 Introduction to Low Shear 101
4.3.2 Hydro-Hermetic Feed Design 101
4.3.3 Power Loss 102
4.3.4 Feed Acceleration Visual and Quantitative
Testing 104
4.3.5 Improved Feed Accelerator 108
4.4 Other Considerations 111
4.4.1 Materials of Construction 111
4.4.2 Clean-in-Place 112
4.4.3 Sterilization-in-Place 113
4.4.4 Containment 114
4.4.5 Surface Finish 114
4.4.6 Temperature Control 115
4.4.7 Water Requirements 115
4.4.8 Noise Level 115
4.4.9 Explosion Proof Design 115
4.5 Examples of Commercial Disk-Stack Centrifuge 115
4.6 Summary 119
References 119
Problems 120
5 Decanter Centrifuge 121

5.1 Solid Bowl or Decanter Centrifuge 121
5.2 Feed Rate 122
5.3 Pool Depth 122
5.4 Rotation Speed and G-Force 123
viii CONTENTS
5.5 Differential Speed 124

5.6 Sedimentation Enhancement Using Chemical 127
5.7 Three-Phase Separation 127
5.8 Cake Conveyance 129
5.8.1 Dry Beach 129
5.8.2 Hydraulic Assist 130
5.9 Summary 132
References 132
Problems 133
6 Commercial Applications of Centrifugation in

Biotechnology 135
6.1 Generic Flow Sheet of Biopharmaceutical 136
6.2 Mammalian Cell 138
6.3 Yeast Processing 141
6.4 Hormones Processing 144
6.5 Insulin Production 145
6.6 Biotech Separation of Inclusion Bodies 145
6.7 Vaccines Processing 147
6.7.1 Concentrated Cell-Based Product 147
6.7.2 Serum Product 148
6.8 Enzymes Processing 148
6.8.1 Extracellular Enzymes 148
6.8.2 Intracellular Enzymes 149
6.9 Probiotic Processing 150
6.10 Aquaculture 151
6.11 Alternative Meat 152
6.12 Baker Yeast Processing 153
6.13 Omega-3 From Microalgae 154
6.14 Ethanol Production 155
6.15 Other Biotech Processing 157
6.15.1 Recovery of Coagulation Factors From
Blood Plasma 157
6.15.2 Tissue From Animal Cells 157
6.15.3 Laboratory Concentration and Buffer
Exchange Using Centrifugal Filter 158
6.16 Summary 159
References 159
Problems 160
CONTENTS ix
7 Concentrating Solids by Centrifugation 161

7.1 Introduction 161
7.2 Concentrating Underflow 161
7.3 Compaction 163
7.4 Expression or Percolation 164
7.5 Compaction Testing 166
7.6 Compaction Pressure 167
7.6.1 Test-Tube Compaction 168
7.6.2 Decanter Compaction 168
7.6.3 Considerations of Cake Compaction 170
7.7 Recommendations for Increasing Solid Concentration
in Underflow 170
7.8 Summary 171
References 171
Problems 172
8 Laboratory and Pilot Testing 173

8.1 Process Objectives 173
8.2 Solid, Liquid, and Suspension Properties 174
8.2.1 Solids Properties 174
8.2.2 Mother Liquid Properties 175
8.2.3 Feed Slurry Properties 175
8.3 Bench-Scale Testing 175
8.3.1 Separability 175
8.3.2 Flocculant and Coagulant in Bench Tests 176
8.3.3 Test Variables 177
8.3.4 Material Balance 177
8.3.5 Acceleration and Deceleration Time Duration 180
8.3.6 Settling Velocity 180
8.4 Centrifugal Filter Testing 185
8.4.1 Steady-State Membrane Centrifugal Filtration
to Determine Protein Diffusivity and Solubility 185
8.4.2 Transient Membrane Centrifugal Filtration to
Determine Protein Osmotic Pressure and
Membrane Resistance 186
8.5 Pilot Testing 187
8.5.1 Material Balance Consideration for Pilot/
Production Scale 188
8.5.2 Product (Protein) Yield 190
8.5.3 Pilot Test Factors 192
8.6 Summary 199
References 199
Problems 200
x CONTENTS
9 Selection and Sizing of Centrifuges 203

9.1 Selection 203
9.1.1 Introduction 203
9.1.2 Tubular Centrifuge Selection 204
9.1.3 Disk Centrifuge Selection 204
9.1.4 Centrifuge Comparison 205
9.2 Centrifuge Sizing 207
9.2.1 Sizes and Rates 207
9.2.2 Dimensionless Le Number 208
9.2.3 Spintube (Bottle) Centrifuge 210
9.2.4 Sizing for Disk Centrifuge 213
9.2.5 Sizing for Tubular, Chamber, and Decanter
Centrifuge 219
9.3 Feed Particle Size Distribution 222
9.4 Performance of Tubular Centrifuge 223
9.5 Summary 224
References 224
Further Reading 225
Problems 225
10 Troubleshoot and Optimization 229

10.1 Troubleshooting 229
10.1.1 Timescale of Occurrence 229
10.1.2 Mechanical or Process Problem 230
10.1.3 Process Problems 230
10.1.4 Mechanical Problem 233
10.2 Optimization 235
10.2.1 Separation Metrics 235
10.2.2 Monitored Variables 236
10.2.3 Controlled Variables 237
10.2.4 Simple Optimization Scheme 237
10.3 Summary 240
Problems 240
11 Visualization and Modeling of Flow and Separation in

Tubular Centrifuge 243
11.1 Flow Visualization 243
11.2 Improved Moving Layer Flow Model 248
11.3 Effect of Velocity Profile 251
11.4 Effect of Friction Within the Flow Layer 252
CONTENTS xi
11.5 Dimensionless Le Parameter 252

11.6 Quantitative Prediction 253
11.6.1 Total Solids Recovery in Cake 253
11.6.2 Total Solids Recovery in the Centrate 253
11.6.3 Particle Size Distribution of Supernatant/
Overflow 254
11.6.4 Cumulative Size Recovery 254
11.7 Sedimentation Tests 255
11.7.1 Experiments on Sedimentation in Rotating
Bowl Centrifuge 255
11.8 Summary 258
References 259
Problems 259
12 Disk-Stack Modeling 261

12.1 Disk Model 261
12.1.1 Continuum Phase 264
12.1.2 Dispersed Phase 264
12.2 Model Validation 268
12.3 Complications 270
12.4 Summary 271
References 271
Problems 272
13 Performance Projection of Centrifuges in Bioseparation 273

13.1 Disk Centrifuge 273
13.1.1 Baseline Case (400-mm Disk) 275
13.1.2 Effect of Fine Size Distribution
(400-mm Disk) 276
13.1.3 Effect of G-Force (580-mm disk) 277
13.1.4 Efficiency η in Le Number (580-mm Disk) 280
13.1.5 Disk Centrifuge for Yeast Processing
(500-mm Disk) 282
13.1.6 Disk Centrifuge for Inclusion Body
Separation (260-mm Disk) 284
13.1.7 Enzymes (580-mm Disk) 286
13.2 Tubular Centrifuge 288
13.2.1 High-G Tubular (150- and 300-mm Tubular) 289
13.2.2 Low-G Tubular (150- and 300-mm Tubular) 290
13.3 Decanter 292
xii CONTENTS
13.4 Spintube 294

13.5 Further Discussion on Numerical Simulations 296
13.6 Summary 297
References 297
Problems 297
14 Rotating Membrane in Bioseparation 299

14.1 Membrane 299
14.1.1 Osmotic Pressure Resistance 300
14.1.2 Gel Resistance 301
14.1.3 Membrane Fouling and Cake Formation 302
14.1.4 Two Scenarios on Rotation 302
14.2 Rotating Disk Membrane With Surface Parallel to
the G-Force 303
14.2.1 Dimensionless Numbers 304
14.2.2 Governing Equations and Solutions 306
14.2.3 Gel Concentration 310
14.2.4 Determining Diffusivity 311
14.2.5 Parametric Effects 313
14.3 Rotating Membrane With Membrane Perpendicular
to the G-Force 315
14.3.1 Spintube Equipped With Membrane
Module—Centrifugal Filter 316
14.3.2 Model on Swinging Bucket Equipped With
Ultrafiltration Membrane 320
14.3.3 Comparing Test Results With Predictions 323
14.4 Summary 328
References 329
Problems 329
15 Flocculation With Decanter Centrifuges 331

15.1.1 Coagulation and Flocculation 331
15.1.2 Decanter Centrifuge 332
15.1.3 Problems 332
15.2 Monotonic Size Distribution Model 333
15.2.1 Moving Layer 334
15.2.2 Floc Model 336
15.2.3 Exponential Floc Size Distribution 336
15.2.4 Leung Number Calculation 338
15.2.5 Model Solution 339
CONTENTS xiii
15.3 Field Test 340

15.3.1 Two Decanter Tests at Wastewater
Treatment 340
15.3.2 Determining In Situ Floc Size 341
15.4 Prediction 346
15.5 Scale-Up 346
15.5.1 Le scale-Up 346
15.5.2 Sigma Scale-Up 348
15.5.3 G/g-Volume Scale-Up 348
15.5.4 Surface Area Scale-Up 348
15.6 Summary 350
References 350
Further Reading 351
Problems 351
16 Case Studies of Monotonic and Unimodal Size

Distribution Models 353
16.2 Monotonic Model Equivalent for Disk-Stack and
Tubular Centrifuges 354
16.3 Disk-Stack Centrifuge for Processing Protein From
Mammalian Cell Culture 356
16.3.1 Low Cell Density Cell Culture 358
16.3.2 High Cell Density Cell Culture 361
16.3.3 Centrate Solids and Turbidity 363
16.4 Tubular Centrifuge for Separating E. coli Lysate 364
16.5 Tubular Centrifuge for Separating S. pneumoniae
Flocculate 366
16.6 Unimodal Size Distribution Model 367
16.6.1 Unflocculated Suspension 369
16.6.2 Flocculation in Disk-Stack Centrifuge 370
16.7 Comparing the Solids Recovery Between Monotonic
and Unimodal Size Distributions 375
16.8 Summary 377
References 378
Problems 378
17 Classifying Bimodal Particle Size Distribution and Case

Study of Inclusion Body Classification 381
17.2 Mammalian Cells With Cell Debris 383
xiv CONTENTS
17.3 Processing Hybridoma Cell Broth 384

17.4 Bimodal Size Model 387
17.5 Application of Bimodal Model on Hybridoma Cell
Separation 388
17.6 Variation in Fine Fractions From Debris 390
17.7 Size of Whole Cells 391
17.7.1 Whole Cells Average Size 392
17.7.2 Size Range on Whole Cells 392
17.8 Effect of Smaller Size (The Debris) 394
17.9 Size Recoveries 398
17.9.1 Size Recovery of Small (S) Particles in
Centrate 399
17.9.2 Size Recovery of Large (L) Particles in
Centrate 400
17.9.3 Example on Classification 401
17.9.4 Smaller Size Fraction Further Apart From
Larger Size Fraction in Bimodal Feed 405
17.9.5 Smaller Size Fraction Closer to the Larger
Size Fraction in Bimodal Feed 407
17.10 Classification of Inclusion Bodies 408
17.10.1 Conventional Inclusion Bodies Processing 409
17.10.2 New Inclusion Bodies Processing 411
17.11 Centrifuges for Inclusion Bodies processing 413
17.11.1 Disk-Stack Centrifuge 413
17.11.2 Tubular Centrifuge 415
17.11.3 Spintube Centrifuge 417
17.12 Separation by Size and Density Difference 418
17.13 Summary 422
References 422
Problems 423
18 Integration of Unified Modeling With Practice in

Centrifugal Separations 425
18.2 Unified Modeling to Centrifugal Separation 425
18.3 Applications of the Unified Separation Models 428
18.3.1 Analysis of Test Data 428
18.3.2 Prediction/Forecast 429
18.3.3 Guiding Testing 429
18.3.4 Optimization 429
CONTENTS xv
18.3.5 Troubleshooting 429

18.3.6 Scale-Up/Scale-Down 430
18.4 Integration of Unified Separation Models With
Practice 431
18.5 Summary 433
Appendix A: Nomenclature 435

Appendix B: Buckingham-π Analysis for Decanter and
Tubular, Disk-Stack, and Spintube Centrifuges 439
Appendix C: Centrate or Concentrate Discharge Through
Rotating Impeller 445
Appendix D: Answers to Problems in Chapters 2 17 449
Index 457
In God, I trust
xvii
Preface to Second Edition (2019)
Since the Centrifugal Separation in Biotechnology (CSB) has been

published in 2007, it has well served the community of biotechnology
separation. I have been contacted by practitioners, researchers, engineers,
students, users, and equipment manufacturers in the biotechnology and
biopharmaceutical community, who told me that they have recommended
the text whole-heartedly to others as they find the contents in it are informa-
tive and helpful. They said CSB is the most comprehensive treatise in deal-
ing with centrifugal separation in biotechnology. There is a good balance of
practice and fundamentals on the subject matter in the text. The text is well
illustrated with tables, figures, sketches, and photographs, and is written in
layman’s language that is easy to understand. The readers also found the
questions at the end of each chapter both challenging and stimulating,
which help them to think and digest the contents that have been discussed.
Over a decade since the first publication of CSB, there has been an
exponential growth in biotech activities. This is especially for the world
of biopharma. As an example, there are much more therapeutic mono-
clonal antibodies that have been under various stages of testing than
before and they have been approved, in record numbers, by the
European Medicines Agencies and the United States Federal Drug
Administration. There is a popular demand from the biotechnology and
biopharmaceutical community for a revised edition of the text. After a
year and a half of preparation we finally come up with the second edi-
tion of the book. We have kept the same approach as our first edition,
again with good “balance” between practice and fundamentals, together
with the author’s 44 years of experience in separation and filtration from
both industry and academia injecting into this delicate balance. We have
updated the text with new technologies and new designs for both the
disk-stack and tubular centrifuges, which are the workhorse of the indus-
try. A decade ago, flocculation was unheard of for disk-stack centrifuge.
Given the bottom feed design which is available today, flocculation can
be implemented for disk-stack centrifuge, when process permits. In
some applications processing flowable concentrate containing biological
cells or bacteria, after separation the concentrate is discharged through
external nozzles. The impact of the concentrate discharge stream at high
speed from the nozzle disk-stack centrifuge results in destroying the
cells or bacteria and losing their probiotic function. Today this probiotic
xix
xx PREFACE TO SECOND EDITION (2019)
flowable concentrate stream can be routed to a small diameter near the

axis of the centrifuge, where the concentrate stream can be gently dis-
charged under pressure without being exposed to air causing oxidation
and being destroyed from high-speed impact. These are only a few of the
new technologies that are presented in the second edition. We have
expanded the discussion on clean-in-place (CIP) and sterilization-in-place
(SIP), which are very important practice for biotechnology and biophar-
maceutical to avoid cross-contamination of their products. Despite there is
no satisfactory standard to execute CIP and SIP, the text has discussed
good practice that can be used as a general guideline for practitioners.
Chapter 6, Commercial Applications of Centrifugation in Biotechnology,
discusses several typical flow sheets for biotech and biopharma processing,
and this has received very favorable response from the readers. We have
included a few more typical flow sheets in biotech and biopharma proces-
sings, and they are by no means exhaustive but serve as good examples.
Some of the flow sheets are related to separation of recombinant protein,
while others are for different applications. Not only have recombinant pro-
tein being used for diagnostic, therapeutics, and disease prevention, they
have been expanded into human food (beef, pork, and chicken made from
recombinant protein) and aquaculture. As medical scientists discover new
antigens that are specific to different types of cancer cells, more monoclo-
nal antibodies are also discovered for identifying the antigens associated
with these cancer cells so that human immune system can launch self-
defense against them. Despite each type of protein being secreted by the
common hosts (mammalian cells, microbial, yeast, virus infected insect
cells, etc.) are different, there are certain commonalities in the separation
and purification steps. CSB continues to use case studies throughout to
illustrate these commonalities so that biotech practitioner not only can use
them in generic form but tailor-make for their own process.
In the first edition, the author has developed basic models of separa-
tion for tubular/decanter and disk-stack centrifuges (Chapter 11:
Visualization and Modeling of Flow and Separation in Tubular
Centrifuge, and Chapter 12: Disk-Stack Modeling). These models have
been used to complement with case studies to discuss separation
(Chapter 13: Performance Projection of Centrifuges in Bioseparation).
One shortcoming is that the particle size distributions, which were used
in Chapter 13, Performance Projection of Centrifuges in Bioseparation,
were often lacking. To that end, some of the models being developed
may be handicapped in short of that measurement. In the second edition,
we have come up with three analytical forms of particle size distribution
for the feed to the separating centrifuge, which depend on two to five
PREFACE TO SECOND EDITION (2019)
parameters. These analytical forms include monotonic size distribution,

unimodal size distribution, and bimodal size distribution. By modifying
the parameters, we can easily change the particle size of the feed and
investigate the sensitivity of the separation outcome from the centrifuge.
We have used these size distributions together with a unified approach
on separation using spintube, disk-stack, tubular, and decanter centrifuges
to study a lot more cases. This includes a very important application on
inferring the flocculated size in centrifuge (Chapter 15: Flocculation With
Decanter Centrifuges), which has been a culprit on scale-up/scale-down
problems. We have also used the monotonic size distribution to interpret
tests being conducted on disk-stack and tubular centrifuges (Chapter 16:
Case Studies of Monotonic and Unimodal Size Distribution Models) with
both dispersed and flocculated feeds, respectively, and to demonstrate the
benefits of flocculation with disk-stack centrifuge (Chapter 16: Case
Studies of Monotonic and Unimodal Size Distribution Models).
Next, we have adopted the bimodal model to classify biologics with
both a smaller and a larger size fraction (Chapter 17: Classifying Bimodal
Particle Size Distribution and Case Study of Inclusion Body Classification)
in the feed to the centrifuge. The size recovery of the smaller fraction, as
well as the larger fraction, in centrate and concentrate, respectively, have
been developed. One can fine-tune the classification in accordance to the
S-shaped size recovery curves to maximize recovery of smaller/larger size
fraction with minimum cross contamination. This can speed-up the devel-
opment or optimization process improving the purity of the products.
Finally, in Chapter 18, Integration of Unified Modeling With Practice
in Centrifugal Separation, we propose an integrated approach, irrespec-
tive of the type of centrifuges, to separation by sedimentation in using
known/measured, or estimated analytical representation of, particle size
distribution to tackle separation problem. The author has suggested inte-
grating the models into practices at all stages from laboratory testing, pilot
testing, and clinical manufacturing to full-scale production. This reassures
the reliability of the separation process despite frequently we have only
limited test results as a basis to make important decisionon the process.
Overall, the second edition not only provides an update to various
centrifugal technologies for biotechnology, but fills in a number of miss-
ing gaps. I believe this new edition of CSB will provide a more power-
ful resource for readers to tackle their centrifugal separation problem.
I sincerely hope that the text can help to push the biotech and biopharma
forward with good practice and advanced technologies in centrifugal
separation. Consequently, drugs substances and intermediates, diagnostic
and therapeutic protein-based drugs, and health supplements, based on
xxii PREFACE TO SECOND EDITION (2019)
recombinant protein and other biotech processes, becomes available in

shorter development time and become more affordable to the general
public.
Finally, I give thanks to all those who have provided valuable inputs
into this new edition. I also thank my wife, Stella, in giving me the
extra-time and space to devote in preparation of the new edition of CSB
that has taken much longer time than I originally estimated. The support
from the rest of my family, my mom, Jessica, Daniel, Jeffrey, and
Sandar has been tremendous. I am grateful to God for His keeping and
strengthening of me, without which I cannot complete this revision.
In Him, I trust.

July 2019
Preface to First Edition (2007)
In processing biological materials to produce high value-added

intermediates or finished products, no matter whether it is liquid or
solid, often involves a separation step, especially after the fermenter or
bioreactor. In one instance, the high-value solid products in a dilute con-
centration have to be separated from the waste liquid with spent cells
and debris; therefore it is essential to prevent loss of valuable solids in
the liquid stream. A further requirement is that contaminants have to be
washed from the solids. Alternatively, the high-value liquid containing
dissolved protein needs to be separated from the biomass and that the
liquid product should be free of solid particles to avoid downstream sepa-
ration and contamination of purification equipment, such as chromatogra-
phy column. The difficulty in carrying out separation step is that biosolids
do not filter well and often foul and blind the filter, such as microfiltration
and ultrafiltration membranes. Instead of filtration, separation by sedimen-
tation utilizing the density difference between the solid and the suspending
liquid can be employed. However, the density difference between bioso-
lids and liquid (typically water based) is very small, rendering the separa-
tion very slow and ineffective, especially under the Earth’s gravity. In
addition, if RNA and other protein materials are dissolved in the liquid,
the liquid phase can be very viscous, which further slows down sedimenta-
tion. Another difficulty is that the solids concentration in suspension for
processing is relatively dilute and requires equipment that has large volu-
metric capacity for handling the flow and process.
Centrifugation has proven to be a rather robust process for enhancing
settling by using thousands to almost millions of times the Earth’s gravita-
tional acceleration. In biopharmaceutical processing for producing a
recombinant therapeutic protein for antibiotics and drug substances from
yeast, microbial, and mammalian cells, such as the Chinese Hamster
Ovary cells, centrifuges have been widely used to perform separation,
classification of cell debris, concentration of suspension, and separation
and washing of solids, such as inclusion body or crystalline protein. No
doubt, given the escalating research activities in biotechnology, many new
sources of therapeutic proteins and other valuable biological materials
will be discovered and developed, and more stringent requirements are
demanded from separation/recovery and purification. There will be more
xxiii
xxiv PREFACE TO FIRST EDITION (2007)
growing needs of centrifugation in combination with other separations and

filtrations to perform the often overlooked, yet important, duty.
While there are many texts and reference books on bioseparation,
there is very little coverage on centrifugation. This book is the first ref-
erence book of its kind devoted to centrifugal separation in biotechnol-
ogy. It is an outgrowth of a series of seminars, short courses and
presentations that the author has delivered to biopharmaceutical compa-
nies all over the world. This new and challenging topic has received
excellent global reception, which is quite comforting and rewarding. The
contents of this book are also based on the author’s research and exten-
sive experiences, respectively, in practice, mentoring and lecturing on
the subject for over 20 years.
The book starts out with an introduction on the topic (Chapter 1) fol-
lowed by Chapter 2 on sedimentation, which is the key step of separation.
Subsequently, various batch (spintube centrifuge, ultracentrifuge) and
semi-batch (tubular centrifuge) centrifuges are discussed in Chapter 3. The
workhorse of the industrial separation process, disk-stack centrifuge, is
presented and discussed in detail in Chapter 4. Also, decanter centrifuge,
which is more applicable to high-solids feed under relatively lower centrif-
ugal acceleration, has also been included in Chapter 5. However, the dis-
cussion will be brief in favor of giving room to various other topics.
Commercial applications of centrifuges in biotechnology are discussed in
Chapter 6. This is perhaps one of the most interesting topics for practi-
tioners who are more concerned about where proven processes are and
how their new process may build on what is already known and practiced.
Despite there being lot of applications discussed in this chapter, unfortu-
nately there might be applications that have been inadvertently omitted,
given that the biotech applications are very diverse. Subsequently, we dis-
cuss in Chapter 7 the importance and practice of increasing, or at least
maintaining, high-solids concentration in the underflow stream of the cen-
trifuge. Laboratory and pilot testing and selection and sizing are essential
functions for establishing and implementing the biotech process and they
are discussed in Chapters 8 and 9, respectively. A new unified approach in
scale-up and prediction with use of a dimensionless Leung (Le) number is
introduced. The Le number works for all types of centrifuges, including
spintube, tubular, chamber, disk-stack, and decanter centrifuges. This pro-
vides a solid foundation for practitioners to scale-up equipment and analyze
test results. Troubleshooting and optimization are two important topics of
general interest, especially for installed machines, and these are discussed in
Chapter 10. Subsequently, modeling of tubular and disk-stack centrifuges
are covered in Chapters 11 and 12, respectively, for researchers who are
PREFACE TO FIRST EDITION (2007)
interested. Readers who are not interested in modeling can jump directly
to Chapter 13. The Le number provides a basis for the scale-up and per-
formance prediction covered in Chapter 13. Here, numerous examples
are used to demonstrate the versatility of the numerical simulator built on
the Le-approach to forecast performance in parallel with concurrent test-
ing, which is often limited for various reasons. Numerical simulation can
also be used to analyze laboratory, pilot, and production test results to
validate machine and process performance. Therefore numerical simula-
tion can be used for laboratory screening, pilot testing, clinical
manufacturing testing, full-scale production testing, and even for small-
scale testing in the laboratory to investigate alternatives and improve-
ments of the existing process under production. Membrane processes,
such as microfiltration, ultrafiltration, and diafiltration, are frequently
used in bioseparation. Lastly, Chapter 14 is devoted to combining two
separation processes: centrifugation and membrane separation. Two
examples, respectively, on centrifugal filter in spintube and large rotating
membrane systems are discussed. The general approach can be extended
to other rotating membrane geometry.
Centrifugation has been treated as a black box in the past, as the sub-
ject is quite complex and nonintuitive. The subject involves multiple dis-
ciplines, such as fluid dynamics, mechanics and vibration, design,
material science, rheology, chemical and process engineering, chemistry,
biology, and physics. I hope this text will fulfill the quest of knowledge
rendering centrifuge a lot more “transparent” to biologists, biotechnolo-
gists, chemists, physicists, scientists, researchers, and practicing engi-
neers. The more they know the better they can deploy, comfortably and
without reservation, centrifuge as handy process equipment.
Problems are listed at the end of each chapter in the text, and they
complement and supplement the contents in the chapter. They are also
meant to reinforce the concept for the readers through practices, chal-
lenging their thoughts and understanding on the topic. Apart from practi-
tioners and researchers, this book is written primarily for 4th year
university student in the 4-year undergraduate study and research gradu-
ates in their MS or PhD research program in university taking biosepara-
tion, bioprocessing, advanced unit-operation/process engineering, or
similar courses.
I am grateful to Stella, Jessica, Jeffrey, my mother, and my late father
for putting up with me while I was devoted to preparing this book. Both
my late father and my dear friend and mentor, the late Professor Ascher
H. Shapiro, demonstrated dedication and perseverance in their lives,
which inspired me all along, especially during the trying times when I
xxvi PREFACE TO FIRST EDITION (2007)
was working on the manuscript among other responsibilities that also

demanded my undivided attention. I also thank Alice Tang for skillfully
helping out with the manuscript work and meeting the publisher’s
deadline.

2007
1
Introduction
1.1 Introduction
Biotechnology has revolutionized our life in the 20th century [1]. Its
impact is only now being felt from engineering food [2 4], engineering
and delivering drugs [5,6], to engineering consumable products. Without
doubt it will continue to influence our daily lives for years to come. One
of the many successful examples is that drugs, such as monoclonal anti-
bodies (mAbs) and some basic drug substances (i.e., the building block
for various drugs), can now be manufactured and formulated from bior-
eaction. One of the commonly used methods in biotechnology is the
recombinant DNA technique [7 9]. A desired gene is isolated from one
organism, and this is inserted into a small piece of carrier DNA called a
vector. It is highly desirable that the recombined DNA (vector plus
gene) can propagate in a similar or unrelated host/recipient cell.
The mammalian cell, such as the Chinese Hamster Ovary cell, is a
popular host cell. Fig. 1.1 shows a schematic of an animal cell which is
very similar to that of a mammalian cell. A characteristic size of the
mammalian cell is about 10 20 µm. Unlike a plant cell, there is no cell
wall for animal and mammalian cells, so they rely on a plasma mem-
brane to keep the intracellular contents intact. High shear stress acting
on the cell can rupture the fragile membrane releasing the intracellular
material.
Yeast (see schematic in Fig. 1.2), in eukaryotic single-celled microor-
ganisms classified as a member of fungus kingdom, has been commonly
used as a host cell in the recombinant DNA process, the knowledge and
experience of which we have gained from the brewery industry. Unlike
a mammalian cell, the yeast cell has a strong cell wall. Yeast cells are
smaller than mammalian cells and are typically between 7 and 10 µm.
Some common yeast hosts include, Saccharomyces cerevisiae (referred
commonly as baker yeast) and Pichia pastoris.
Bacteria, such as Escherichia coli (hereafter abbreviated as E. coli)
and Bacillus subtilis, have been used as host cells for the recombinant
Centrifugal Separations in Biotechnology. DOI: https://doi.org/10.1016/B978-0-08-102634-2.00001-5

© 2020 Elsevier Ltd. All rights reserved. 1
Figure 1.1 Animal cell schematic showing plasma membrane.
Figure 1.2 Yeast cell schematic showing both cell wall and membrane.
Introduction 3
Figure 1.3 Escherichia coli cell schematic.
DNA technique. A schematic of E. coli bacteria cell is shown in

Fig. 1.3. Again, E. coli has a sturdy cell wall with both an outer mem-
brane and an inner membrane. E. coli is typically elongated with a
dimension of 3 µm long by 1 µm width. Therapeutic protein can be
“expressed” by these host cells or organisms with the recombinant
DNA. The protein of interest may remain in the cell (intracellular) or be
secreted to the exterior of the cell (extracellular). The aforementioned
biosynthesis provides more engineering flexibility, specificity, versatil-
ity, reliability, and cost-effectiveness.
Therapeutic proteins are quite diverse in the application treatments,
such as human insulin for diabetes, erythropoietin for anemia and chronic
renal failure, interferon-beta and gamma for cancer, DNase for pulmonary
treatment, vaccines for hepatitis B, interleukin-2 for AIDS, prourokinase
for heart attacks, and tissue plasminogen activator (enzyme) for strokes.
Therapeutic proteins are present in many different kinds of mAbs.
mAbs are antibodies that are identical, because they are produced by
one type of immune cells, and they are all copies or clones of a single
parent cell. mAbs are first produced by Kohler and Milstein in 1975 [10],
for which they were awarded the Nobel Prize in Physiology or Medicine
in 1984. By virtue of the mAb being identical copies produced by one
type of immune cells, they have a high specificity for their targets. mAb
has been used in diagnosis. There are over 100 different diagnostic pro-
ducts available in the world that are mAb [11]. mAb is also used for
4 Centrifugal Separations in Biotechnology
therapeutics. For example, mAb has been a popular antibody made in the
laboratory used for cancer treatment where the antibody is designed to
attach as a label to their counterpart protein (antigen) on a specific cancer
cell so that immune cells can spot and attack the cancer cells. As an
example, the mAb known commercially as alemtuzumab drug (note all
mAb drugs have the last three alphabets labeled as “mab,” which distin-
guishes them being mAb-based drugs) can target at the antigen CD52
found on the cancer cells that causes chronic lymphocytic leukemia [12].
As antigens are discovered to be linked to more specific cancers, more
mAbs have also been developed for cancer treatment. Some mAbs work
better on certain cancers than others. mAb can also attach to the antigen
on breast cancer cells blocking the growth of breast cancer cells.
Cancer cells can “turn off the switch” of immune cells to avoid being
attacked by the immune system in our bodies. Inhibitors, or commonly
known as checkpoints, are mAb produced by the recombinant protein
process, that inhibit the protein secreted from the cancer cells in “fool-
ing” the immune cells, thereby allowing the immune cells to carry out
their normal functions. As an example, PD-1 is a protein on the immune
T-cells. The immune T-cells are normally in a switch-off condition
because PD-1 has been attached by their counterpart PD-L1, another
protein that both normal cells and cancer cells have. In other words,
they have been switched off. Some cancer cells have an abundant PD-
L1 that is used to attach to the PD-1 of the immune T-cells thereby
evading being attacked. On the other hand, mAb can target at either PD-
1 or PD-L1 and inhibit their binding, thereby allowing the immune cells
to attack the cancer cells. For example, pembrolizumab is a PD-1 inhibi-
tor that can treat skin melanoma, non-small-cell lung cancer, kidney can-
cer, bladder cancer, head and neck cancer, and Hodhkin lymphoma [12].
As another example, atezolizumab is a PD-L1 inhibitor that can treat
bladder cancer, non-small-cell lung cancer, and Merkel cell carcinoma
[12]. New inhibitors are being developed rapidly over time as more
knowledge is being gained on the specifics of different cancers and their
behavior. The examples mentioned in the forgoing are just a few under
the broad umbrella of immunotherapy for which mAb plays an impor-
tant role. The main objective of immunotherapy is to enable the immune
system of patients to recognize or target specific cancer cells and destroy
them [13]. In 2005 the total mAb therapeutics entering first-in-human
studies per year is 35, 16 out of which are for cancer treatment. In 2017
the total mAb therapeutics rose to 105, and nearly 80 were for cancer
treatment. Indeed, the antibody therapeutics entering clinical study and
being approved are in record numbers [14].
Introduction 5
Extracellular proteins secreted from yeasts are produced for making

insulin, human serum albumin, and hepatitis vaccines. Insulin drug has
reached over USD 24 billion market in 2018 according to a market study
in 2019. The fast-growing biopharmaceutical business in producing ther-
apeutic proteins is getting so popular that all major drug manufacturers
also carry a parallel line of this business.
Unfortunately, the protein expressed from the bioprocess is in very
small amounts in a large volume of suspension, that is, low concentration.
The two key hurdles in recombinant DNA techniques to produce therapeu-
tic protein [15] are (a) to recover this small concentration of protein after
fermentation by separation and (2) to provide high purity of the protein
product through separation and purification. It is prudent that both separa-
tion and purification processes should be robust and cost-effective for the
biopharmaceutical technology to be viable and competitive. Although this
text is focused on separation, one should bear in mind that given these two
steps are sequential, poor separation can adversely affect purification
downstream. Therefore it is prudent to have an integrated approach for
downstream processing. To say the least, if there is an upset from the fer-
menter upstream producing, say, off-spec finer feed, the centrifuge should
take on the upset feed and try to produce a consistent output downstream
to the filter, membrane and chromatography column downstream in the
interim, while the upset condition is being fixed. Otherwise, the entire
chain of downstream processes can be seriously affected.
Other biotechnology involves synthesis and/or modification of inter-
mediates or final products. Frequently, this is in a suspension form so
that mechanical mixing, separation, spray or thermal drying, and other
allied processes are required.
Given separation is an important task [16 20] in biotechnology in
lieu of the above, it can be a very difficult task due to the low concen-
tration of the protein present and the large volume of liquid to handle,
the fragility of the cells, the presence of cell debris, fine particulates and
colloids, and the high viscosity due to dissolution of intracellular sub-
stances, such as RNA. Typically, separation can be achieved by filtration
and sedimentation. There are some specific problems relating to each as
discussed in the following.
Filtering a suspension containing biomass is quite tricky as the mate-
rial can foul the filter surface, reducing permeate or filtrate flow regard-
less whether the media is a microfiltration or an ultrafiltration
membrane. It is equally challenging to settle biomass as the density of
the biomass material is just slightly greater than that of the liquid phase,
which often is aqueous based. Given that settling rate is proportional to
the difference in the two densities, it takes a very long time to separate,
translating in simple terms—to an impractically low-capacity, high-cost
operation. On the other hand, separation by sedimentation can be much
enhanced under centrifugal acceleration. This is possible by introducing
the suspension with biomass in a centrifuge rotating at high speed where
centrifugal acceleration can be hundreds to millions of times that of the
Earth’s gravitational acceleration.
1.1.1 Common Host Cells for Secreting Recombinant Protein

Fig. 1.4A shows the use of centrifugation in biopharmaceutical produc-
tion of therapeutic protein using the commonly employed mammalian
cells, bacteria and other microbial hosts [21], and yeasts. Centrifugation
(A)
Fermentation
and bioreaction
Mammalian
cell Bacteria Yeast
Extracellular Intracellular Extracellular
Centrifuge Centrifuge
Lysing
separation clarification
Solid product Liquid product
Depth filter Centrifuge Centrifuge lysate Centrifuge

polishing IBClassification clarification polishing
Centrifuge/filter
IB washing
polishing
Downstream processing
Drug substance and monoclonal antibodies
Figure 1.4A Drug substances produced from fermentation and down-

stream processes where centrifugation has been widely employed for
various duties.
Introduction 7
can be used for separation, clarification/polishing, thickening, classifica-

tion, and washing-and-separation.
With reference to the left bioprocess in Fig. 1.4A, after harvesting
from the bioreactor, the cell culture suspension containing mammalian
cells is sent to a centrifuge wherein the cells are separated from the
liquid product which contains the extracellular protein secreted from the
mammalian cells. The separated liquid is then sent to a depth filter for
further polishing, removing any solid particulates before sending it
downstream for processing. Some mammalian cells also secrete
soluble protein intracellularly, despite majority secrete liquid protein
extracellularly.
With reference to the middle bioprocess in Fig. 1.4A, the protein is
expressed intracellular in the bacteria. After harvesting and homogeniz-
ing, the protein is released from the lysed bacteria in the inclusion
bodies (IBs) that need to be isolated before additional downstream pro-
cessing. Centrifugation is used to sediment the IBs while the cellular
contents, cell debris, and finer materials leave with the liquid phase to
wasting. The IB is further washed and separated several times until it
reaches the desired purity for downstream processing. Alternatively, the
protein from the bacteria may be expressed in the intracellular liquid,
and upon homogenizing, this protein is released in the liquid. The task is
to remove all solid materials and to recover the liquid bearing the solu-
ble protein. The biomass may have to be washed to ensure all the pro-
tein that is adhered to the biomass has been recovered, otherwise this
represents a loss or lower yield for the process.
With reference to the right bioprocess in Fig. 1.4A, after harvesting
from the fermenter, the yeast suspension is sent to centrifugation where
the liquid containing extracellular protein is separated from the yeast
solids. The liquid leaving the centrifuge may have to be centrifuged
again (i.e., clarification or polishing) to remove any particulates and tur-
bidity before downstream processing. Some yeast cells may also secrete
protein intracellularly as liquid protein.
The above three paths are central to biopharmaceutical production of
therapeutic proteins using host cells. These will be discussed in much
greater detail throughout the text.
Besides mammalian cells, microbials, and yeast, infected baculovirus
insect cells can also secrete protein. For example, baculovirus-infected
insect cells can express mAb, such as antibody C017-1A that recognizes
antigen GA733 on colorectal cancer cell. Once the mAb C017-1A is
docked onto the antigen GA733, immune cells can attack the colorectal
cancer cells [22]. They are being used for multiple gene delivery
platforms with subsequent cellular expression of protein for labeling

cells that can serve as sensors for monitoring cellular architecture or
metabolism, and other functions [23].
1.1.2 Platform for Protein Expression

Despite we have used as an introduction in Section 1.1.1 the three most
common host cells to discuss the downstream separation and recovery of
protein, it is more prudent to examine how protein is being secreted irre-
spective of the host cells, from which the downstream protein separation
and recovery steps will typically follow.
In general, there are three platforms for recombinant protein expres-
sions from cells and microorganisms. The protein can be secreted extra-
cellularly into the broth of the cell culture in the bioreactor or fermenter.
It can also be secreted inside the cells in a bioreactor or fermenter.
There are two types of intracellular expressions. In one type, the protein
is secreted inside the cell as liquid form. In another type, the protein is
secreted as a dense mass, commonly referred as IB, which contains mis-
folded and biological inactive protein. This is illustrated in Fig. 1.4B. It
is possible that some cells or microorganisms secrete mostly intracellular
protein but also a small amount of extracellular protein and vice versa.
Mammalian cells have been thought of secreting only extracellular pro-
tein, but some new processes and host cells have shown that they can
also secrete intracellular protein. They can release the protein in the
(B) Recombinant
protein expression
Extracellular Intracellular Intracellular

protein (liquid) protein protein
(liquid) (inclusion
Protein bodies)
IB
Protein
Cell Cell
Cell
Figure 1.4B Three types of secreted recombinant protein by cells and

microorganisms.
Introduction 9
broth during maturity without the need of cell lysis. With the advent of
new research and technology, it is expected that more new recombinant
processes and new host cells will be discovered over time. Regardless,
we can look at these protein expression processes being the “model” for
downstream processes, including separation, which is a key step
upstream of purification, whether it be extracellular or intracellular pro-
tein or even a hybrid of both. Without achieving the objective in separa-
tion, the steps downstream of separation will only become more difficult
leading to poor protein recovery and low yield. Below, we will briefly
describe each of these expression processes and the downstream flow
sheet in general terms.
1.1.3 Extracellular Protein

Protein is secreted by the mammalian cells in cell culture or yeast in
fermenter extracellularly. When protein mixed with the broth is har-
vested from the bioreactor or fermenter, the objective is to remove any
cells or debris from the broth as the liquid contains the liquid protein. If
centrifugation is used to carry out the separation, the liquid product
should be clarified (free of suspended cells and cell debris) while all the
solids should settle in the concentrate stream. The centrate product will
be subsequently sent to the depth filter or microfiltration for polishing,
that is, to remove any residual fine solids not being removed by centrifu-
gation. Downstream, the liquid product is washed (reslurry with buffer
solution) by diafiltration to dissolve any dissolved impurities, including
salts, and subsequently sent to ultrafiltration for concentration. Finally,
the protein is purified in the chromatography column. The process flow
sheet is shown in Fig. 1.4C.
1.1.4 Intracellular Protein—Liquid

Protein can be secreted inside the cells using mammalian cells, yeast,
and bacteria in the bioreactor or fermenter. For yeast and bacteria, at
harvest, the cells are lysed breaking the cell walls releasing the protein
liquid that will be mixed with the broth. The objective of the separation
by centrifugation subsequent to lysis is to remove the cell debris and
some unlysed cells from the protein liquid. The liquid lysate is clarified
using centrifuge in this step. Subsequently, the liquid protein will go
through fine filtration, washing, and concentration, the steps of which
are analogous to the case of extracellular protein. Finally, the protein is
purified by the chromatography. It is perceived that given the size of the
cell debris is very small on the order of submicrons, centrifugation at
(C)
Cell culture/
fermentation
Clarification and
Cell cell separation
(centrifugation)
Protein
For example Fine filtration

mAb, (depth filtration/
enzymes, microfiltration)
antibiotics,
amino acids
Washing and
concentration
(diafiltration,
ultrafiltration)
Purification
(chromatography)
Figure 1.4C Extracellular protein secreted into broth.
high speed is necessary to generate large centrifugal force to carry out

separation. It is indeed a very important step in the process. Otherwise,
any unseparated cell debris will be carried downstream overloading the
depth filter/microfilter and even to the diafilter and ultrafilter and possi-
bly fouling the chromatography column. The process flow sheet is
shown in Fig. 1.4D.
For mammalian cells, there are exceptions for which the intracellu-
lar protein secreted can be released into the broth. Therefore periodi-
cally the broth is removed from the bioreactor, and separation is made
for which the separated whole cells are returned back to the bioreactor.
The product centrate with the cell debris will undergo another separa-
tion, where debris are removed by high-speed centrifugation. The cen-
trate liquid containing protein is sent to the depth filter for further
polishing.
1.1.5 Intracellular Protein—Inclusion Body

Extracellular protein can be secreted quite prolifically from the micro-
bials, commonly bacterial cells, in the production of recombinant pro-
tein. Unfortunately, the protein is misfolded and inactive and requires
Introduction 11
(D)
Fermentation/
cell culture
Cell harvest
Cell lysis
Protein
Cell
Lysate
clarification
For example (centrifugation)
enzymes,
growth,
factors,
vaccines Fine
separation,
washing,
concentration
Purification
(chromatography)
Figure 1.4D Intracellular protein secreted inside the cells.
subsequent processing to restore its bioactivity before use. At harvest, the

cells are lysed releasing the protein in the dense IBs. Subsequently, the IB
cells are homogenized releasing the IB cells. The IB cells have to be sepa-
rated from the cell debris and in order to have effective separation, the
properties for which separation is based should be distinctly different.
Typically, the IB cells are in the size range of 0.2 1.4 µm [24]. The den-
sity of the solids is about 1.3 g/cm3, but the IB cells have large void
fraction filled with liquid; therefore the effective bulk density may still be
about 1.1 g/cm3 or smaller depending on the void fraction. On the other
hand, the cell debris in the lysate is also quite small, 0.5 µm and below,
and has bulk density in the same range as the cell debris. In the separation
step, the IB cells are separated from the cell debris. High-speed centrifu-
gation is used to settle the IB cells from the liquid leaving the fine cell
debris to leave with the liquid centrate from the centrifuge. This fraction-
ation or classification step is not perfect. Given the IB cell size and the
cell debris are not too far from each other, there is always some cell debris
mixed with the IB cells in the sediment. This interesting case will be taken
up in Chapter 17, Classifying Bimodal Particle Size Distribution and Case
Study of Inclusion Body Classification, when we discuss classification.
(E)
Fermentation
IB
Cell harvest
Cell
Cell lysis
Classification
Inclusion bodies and
cell debris
(centrifugation)
Solubilization,
Solubilization Refolding refolding
Purification
(chromatography)
Figure 1.4E Intracellular protein secreted into the inclusion bodies.
The IB cells are washed and centrifuged a few times before sending
to the next process step. There the IB cells are solubilized moderately
by alkaline-based chemicals to dissolve the protein IB. Subsequently the
misfolded protein is refolded to restore the bioactivity of the protein.
The protein in liquid is subsequently purified in chromatography. If clas-
sification of the IB cells and cell debris is not carried out properly by
centrifugation, cell debris will be carried downstream causing contami-
nation and fouling of the chromatography column. On the other hand,
IB cells which have not been separated by centrifugation and leave the
centrate also represent a loss of yield for the entire process. The process
flow sheet is summarized in Fig. 1.4E.
1.2 Centrifugal Separation and Filtration

Industrial centrifugal separation [25,26] can be divided generally into
two classes: sedimenting and filtering. It should be noted that straight
centrifugation can only separate suspended solids and not dissolved
solids with the exception of a centrifugal filter, to be discussed later,
which can separate soluble solids. Heavier solids settle to the solid wall
of a sedimenting centrifuge under centrifugal acceleration that is much
Introduction 13
greater than the Earth’s gravity. A density difference between the solid
and the liquid phase is required to affect separation. A schematic of a
sedimenting solid-wall centrifuge is shown in Fig. 1.5. Similarly, a ligh-
ter dispersed solid phase, like fat or solids with attached air bubbles, can
also float (instead of sink) in a continuous liquid phase, and separation
by flotation can be enhanced in a centrifugal field.
On the other hand, density difference between the two phases is not
required to separate the solid from liquid phase in a filtering centrifuge.
Both phases are driven under the centrifugal body force to the perforated
wall lined with a filter medium. Liquid permeates (see Fig. 1.6) through
the filter medium while solids, comparable or larger than the openings
of the filter medium, are retained. Sometimes even smaller solids can be
retained as they “bridge across” or “jam” the medium openings preclud-
ing them from filtering through. As such, openings in the filter medium
can be selected normally two to three times larger than the particle size
to be retained. Once a “cake” layer of particles forms on the medium
despite some smaller particles that may still percolate through during
initial filtration, the cake layer further acts as a frontline filter medium
in series with the original medium to retain the incoming particles.
Slurry pool
Axis
Solid wall
Cake
Figure 1.5 Solid-wall sedimenting centrifuge.
Cake
Axis Filtrate
Slurry pool Filter medium
Figure 1.6 Filtering perforate-wall centrifuge.

It can be seen that filtering centrifuges can separate particles and liquid
regardless of their density difference; this is very much different from a
sedimenting centrifuge that relies on the density difference of the two
phases to drive separation.
1.2.1 Sedimenting Centrifuge

Sedimenting centrifuge can be divided into, respectively, batch and con-
tinuous sediment discharge as represented in Fig. 1.7. For batch dis-
charge, this can be further divided to batch and continuous feed.
Spintube, ultracentrifuge, zonal centrifuge are all classified as batch feed
centrifuges despite some zonal centrifuges can have continuous feed and
continuous removal features. In batch feed, a fixed amount of feed slurry
is introduced to the centrifuge. Upon separation, heavier solids settle to
the bowl wall or tube bottom at a large radius. Here they accumulate
temporarily until separation stops.
Tubular, manual disk, chamber, multibowl, and solid basket are con-
sidered as semicontinuous as they take continuous feed of suspension.
Heavier and larger solids from suspension get settled under centrifugal
Sedimenting
centrifuge
Batch Continuous
discharge discharge
Continuous
Batch feed Disk Decanter
feed
Dropping- Conventional
Spintube Tubular
bottom disk decanter
Ultra- Manual Nozzle Dual-cone

centrifuge disk disk decanter
Zonal Chamber and Compound-

centrifuge multibowl beach decanter
Solid Nozzle
basket decanter
Figure 1.7 Batch and continuous centrifuge classification.

Introduction 15
acceleration and the sediment is stored temporarily in the bowl until the
quality of the separated liquid gets affected by the growing sediment in
the bowl. At that point, feeding stops, the centrifuge is allowed to coast
down, the liquid pool is drained, and the sediment is removed. The cen-
trifuge needs to be cleaned before the next cycle.
For continuous or semicontinuous discharge of sediment, both disk
and decanter centrifuges fall in this category. Under disk centrifuge,
there are two types depending on how the concentrated solids are dis-
charged—dropping bottom with intermittent solid discharge, and the
nozzle disk with continuous solid discharge. On the other hand, there
are four types under decanter, a conventional decanter, dual-cone
decanter for classifying two solids and a liquid phase, compound beach
decanter for dewatering fine solids producing a paste-like cake [27], and
nozzle decanter for classifying kaolin suspension with 1 2 µm particles.
1.2.2 Filtering Centrifuges

Filtering centrifuges are also divided into two types: batch and continu-
ous feed (see Fig. 1.8). Under batch discharge, there are two categories.
First, a small-batch feed under which perforated spintube and basket and
centrifugal filter both belong. Second, a large-batch feed under which
conventional basket, peelers, siphon, and inverting bag centrifuges all
belong. Regardless of the large- or small-batch feed, they take a batch
of feed suspension or “charge” and perform various operations to pro-
cess the suspension, including filtering, washing, deliquoring/dewatering
with or without drying, unloading and decelerating (for peeler) or decel-
erating and unloading (for regular baskets), and cleaning.
Pusher, conical screen, and screenbowl are continuous centrifuges.
Feed is continuously introduced in these centrifuges, and cake retained
by the filter media is continuously being removed. Filtrate liquid, with
minimal suspended solids, is also removed separately.
Suspension containing biological solids filter very slowly and they
often clog up the filter media, forming an impermeable cake. As a con-
sequence, filtration is often affected by a thin cake filtration in a con-
trolled batch mode under moderate centrifugal gravity so that the cake
does not compact to an impermeable “skin” layer adjacent to the filter
medium. In addition, solids should be at least greater than 10 µm, other-
wise filtration is slow and impractical. If the valuable product is the liq-
uid, it is possible to use a filter aid, such as diatomaceous earth, to
enhance the filtration rate provided the filter aid does not interact with
the product. In essence, this increases the permeability of the filter cake.
Filtering
centrifuge
Batch Continuous
discharge discharge
Small-batch Large-batch Conical

Pusher Screenbowl
feed feed Screen
Perforated Conventional Single-stage Conventional Scroll

spimtube basket pusher screenbowl screen
Peeler and
Centrifugal Multistage Insitu cake Vibrating
siphon
filter pusher washing screen
basket
Inverting-
Tumbling
bag
screen
basket
Wide-angle
screen
Figure 1.8 Batch and continuous filtering centrifuge classification.
When the product protein is in the solids, such as in the biological cells,
filter aid should not be used as it is almost impossible to separate the fil-
ter aid from the valuable biological material.
1.3 Pros and Cons of Filtration Versus

Centrifugation
Centrifugation followed by depth filtration, two-stage depth filtration, or
microfiltration diafiltration can all be used alone for solid liquid sepa-
ration when the valuable protein is in liquid phase. With reference to
Fig. 1.4A, centrifugation is frequently used for lysed cells involving
release of protein solids to be separated from the cell debris when bacte-
ria are used as host cells. Table 1.1 compares the pros and cons of cen-
trifugation and microfiltration. It is quite interesting that centrifugation
Introduction 17
Table 1.1 Comparing centrifugation and microfiltration (MF).
Pros Less shear compared with that generated from tangential

flow filtration or crossflow filtration
Cell debris does not foul or clog up pores leading to blinding;
advantageous for higher feed solids (future trend)
Remove solids down to 0.2 µm at high G
Compact
Less downtime (from fouling)
Single pass or multiple passes
Robust
Cons Slightly higher capital costs
Comparable operating costs (power and maintenance) as MF, which requires

periodic replacement of membranes from fouling.
actually generates less shear stress than tangential flow filtration con-
trary to conventional wisdom, provided a good feed acceleration is
adopted in the centrifuge, see Chapter 4, Disk Centrifuge. Also, centrifu-
gation does not suffer from the fouling of membrane that leads to costly
membrane replacement and downtime. Also, it is a very robust and reli-
able system.
The solids in suspension for the process of interest are very small, in
the domain of 10 µm and below, and sometimes even in the 1 2 2 µm
range. While the small density difference affects sedimentation and not
as much for filtration, as discussed, the fine biosolids affect depth filtra-
tion, clogging flow path of the filter, leading to rapid large pressure drop
across the depth filter. For microfiltration, the membrane needs to be
stayed “unclogged” or “unfouled” by the use of a crossflow using a high
shear rate to scour the membrane surface (see Chapter 14: Rotating
Membrane in Bioseparation) and to use cleaning agent to wash the
membrane during downtime. Also, diafiltration helps to maintain a
lower solid concentration, to prevent fouling. Nevertheless, fouling lead-
ing to the replacement of membrane and an associated downtime are the
key drawbacks of microfiltration in this application. In addition, a much
larger volume of liquid product (two to four times the original volume)
results from washing or diafiltration to recover the protein. This implies
a higher cost for the process as it involves more concentration by ultra-
filtration and other processes downstream for treating the spent liquid.
A fourth possibility is to combine centrifugation with depth
filtration as an integrated approach to separate mammalian cells.
Centrifugation takes up the solids loading from the feed stream leaving
the fermenter/bioreactor, while the depth filter works best in removing

the submicron particles (separated liquid from the centrifuge) from a
low-solid stream leaving the centrifuge. This will be discussed in more
detail in Chapter 6, Commercial Applications of Centrifugation in
Biotechnology.
1.4 Generic Flow Sheet for Biopharmaceutical

Process
The recombinant protein process has become very popular for engineer-
ing a protein to have specific configurations and functions [2 9].
Various recombinant proteins are commonly expressed through an engi-
neering culture such as yeast, bacteria (e.g., E. coli), and living cells
(e.g., mammalian and plant cells). The extracellular (exterior of cells)
protein expressed by yeast and mammalian cells are in the liquid phase,
while protein expressed by the engineered bacteria is inside the bacteria
(i.e., intracellular), which subsequently needs to be lysed to release the
protein. The process condition of a cell culture is carried out under
specific range of temperature, pressure, and agitation/mixing, with fer-
mentation usually subject to shorter time and more intensive condition
(higher process temperature, pressure, and with more rigorous mixing),
while bioreaction takes place under longer reaction time and milder con-
ditions (lower process temperature, pressure, and only moderate mixing).
Fig. 1.9 shows a generic flow sheet for processing recombinant pro-
tein in which protein is expressed extracellularly wherein liquid is the
product. The immediate first step—the separation step—is also referred
to as primary recovery of protein. After solid liquid separation, by any
of the aforementioned four different possible separation methods, the
protein buffer liquid may be replaced or diluted with a more appropriate
buffer with a different pH and ionic strength followed by a concentration
using an ultrafiltration and diafiltration combination. The end product
is a concentrated protein solution in a suitable buffer liquid. At
this stage, the protein can be purified using ion-exchange or affinity
Bioreactor/
cell culture
Concentration,
Purification
Coarse and buffer Sterile Drug
Centrifugation (chromatography)
fine filtration exchange filtration substance
(UF/DF)
Fermenter
Figure 1.9 Generic flow sheet of biopharmaceutical drug substance.

Introduction 19
chromatography to remove any impurities and contaminants. A final

sterile filtration involves the use of a 0.2-µm-sized microfilter to remove
bacteria that are incurred during processing. The final product is typi-
cally a drug substance or an antibiotic.
1.5 Other Centrifugal Separations

Other than for primary recovery in the downstream process of recombinant
protein, centrifugal separation is also used in many biotechnology
solid liquid separations in manufacturing of drugs/hormones such as insu-
lin and many others. In the process of manufacturing, drugs (in solid form)
frequently contain salt and other impurities. In such cases, the drugs need
to be washed by reslurrying followed by centrifugal separation. Also, crys-
tallization and precipitation in the purification step require solid liquid
separation by centrifugation. The objective in these processes is to fully
capture or recover the valuable suspended solids, unlike the recovery of
soluble protein expressed extracellularly by yeast and mammalian cells
wherein the product is the liquid. Another equally important objective is to
reduce the impurity level of the crystals to an acceptable level for down-
stream formulation, such as washing followed by centrifugal separation.
1.6 Inputs and Outputs of Centrifuge

Centrifuge has often been considered as a black box, as the mechanics
and fluid dynamics are quite complex. Here we will discuss the scien-
tific basis and understanding of centrifugation and operation of various
types of available centrifuges as commonly used in separation in bio-
technology. In the simplest terms, for a centrifuge processing, a wet feed
suspension after centrifuging, a centrate, supernatant, or overflow con-
taining a small amount of suspended solids leave the centrifuge together
with a moist concentrate, wet cake, or underflow. This is depicted in
Fig. 1.10. There could be also another input such as chemicals (coagu-
lants and flocculants) added to flocculate the feed suspension (not shown
in Fig. 1.10). Based on the previous discussion, the valuable protein
Centrate/effluent/
Feed Centrifuge overflow liquid
Concentrate/cake/
underflow solids
Figure 1.10 Typical input and output streams of centrifugation.

product can be in the fine suspended solids, such as the IBs, crystals or
precipitants containing protein, or in the centrate liquid phase, as in the
extracellular protein expression (yeast and mammalian cells). The centri-
fuge needs to be tuned to separate the product from the rest (waste or
recycle stream). Depending on the specific process, as discussed below,
some metrics or measures are commonly used to assess the centrifugal
separation.
1.7 Separation Metrics

Several measures of centrifuge performance are common—protein yield,
suspended solids in the centrate or solid recovery, throughput or capacity,
and cell viability.
1.7.1 Protein Yield

For soluble protein expressed from extracellular process, one important
measure of the separation performance of the centrifuge is the protein
yield Y. Yield is defined as the ratio of the amount (e.g., kg/min or
g/min) of protein recovered in the liquid product to the amount (kg/min
or g/min) of protein in the feed to the centrifuge. A complete recovery
of protein without loss is 100%. Usually, the yield should be very high
before the separation process can be considered viable. A 90% or higher
in yield is typical. The specific yield depends on how difficult is the sep-
aration. An example on protein yield is given, respectively, in
Chapter 7, Concentrating Solids by Centrifugation, and Chapter 8,
Laboratory and Pilot Testing.
For continuous feed centrifuge, the volumetric rate (L/min) and protein
concentration of both feed and centrate need to be measured, respectively,
for the calculation of yield. For batch feed centrifuge, the volume and
protein concentration of both feed and supernatant (i.e., centrate) should
be measured, respectively, for the yield calculation. It is evident that liq-
uid loss in the concentrate or cake affects the yield as the protein is dis-
solved in liquid; therefore the amount of liquid in the concentrate should
be minimized (see Chapter 7: Concentrating Solids by Centrifugation).
1.7.2 Centrate Suspended Solids

The centrate suspended solids should be minimized unless this is for
classification, wherein finer-sized solids in the centrate are separated
from larger solids in the concentrate as found for separating cell debris
Introduction 21
from IBs. A measure of clarity of the liquid centrate is the amount of

suspended solids by weight or by bulk volume after the centrate is spun
in a spintube centrifuge for a prescribed time. For cell culture, only fine
solids in the submicron range escape, with the centrate or supernatant to
be ultimately captured by the downstream filter.
An indirect method on assessing the centrate suspended solid is to
measure the optical opacity (or turbidity) of the centrate liquid. The tur-
bidity measurement should be calibrated against a standard on a frequent
basis. A consequence of good clarification is that the solids recovered
by sedimentation (kg/h, dry basis) compared with the feed solids (kg/h,
dry basis) should be very high. The ratio is referred to the solid recovery
Rs. When Rs is at 100%, this implies perfect separation, and there is no
solid in the product centrate or supernatant. For cell culture, we may
achieve, say, 99.9% recovery of cells by centrifugation, leaving minimal
cells escaped in the centrate or supernatant.
1.7.3 Throughput Rate

The volumetric rate or capacity of centrifuge (in L/min) is an important
measure of the volumetric liquid throughput capacity that a centrifuge
can attain. Once the total capacity (size and number) of fermenter or
bioreactor is fixed, the rate of the centrifuge(s) is determined, and this in
turn bears out the total capacity (size and number) of centrifuges. High-
rate larger centrifuges require fewer centrifuges when compared with
low-rate smaller centrifuges. On the other hand, a spare centrifuge needs
to be furnished to cover the operating centrifuges when one of these cen-
trifuges is rotated out for maintenance. Again, the operation planning
requires the information on centrifuge throughput rate.
1.7.4 Cell Viability

Mammalian cells are gaining popularity in expressing protein as there is
more flexibility in pursuing this route. However, unlike plant cells or
yeast, mammalian cells are very shear sensitive as they do not have a
cell wall. They are highly susceptible to shear, such as during accelera-
tion of the feed stream. Cells can be destroyed in the process, releasing
an intracellular protein substance that can be harmful for downstream
purification (cross-contamination of product protein) and finer debris
which renders the separation problem more difficult, resulting in
increased suspended solids loading up the depth filter. As a minimum,
cell viability of mammalian cell lines should be maintained at a high
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looking as we do on Christian there before us, we see that the features of
her brilliant countenance are as like as brothers and sisters may be—like,
and yet unlike, for the pressure of that great sorrow has fallen lightly on
little Mary’s buoyant spirit. She is still “little Mary,” though her head is
higher now than Christian’s, who calls her so. Those two years have added
no less to her inner growth than to her stature, and Mary Melville, with all
the mirth and joyousness of her earlier girlhood, has the cultivated mind of
a woman now. There are many bright young faces shining in this gay room,
but there is not one like little Mary’s; not one eye in this assembly can boast
such a sunny glance as hers, graver than her peers when it is called to look
on serious things, and beaming then with a youthful wisdom, which tells of
holy thoughts and pure intents within, and anon illumined with such a flash
of genuine mirthfulness and innocent gaiety, so fresh and unconscious in its
happy light, as would startle the sternest countenance into an answering
smile. She is much loved, our sprightly Mary, and is the very sun and light
of the circle she moves in; and friends who have known her from her
childhood, tell one another how like she is to Halbert, and shake their
heads, and are thankful that she can never be exposed to similar
temptations. Do they think that Mary, like her brother, would have fallen,
that she must succumb too, before the adversary’s power, if tried as hardly?
Ah, it is not well that the innocent lamb, so tender, so guileless and gentle,
should be exposed to the power of the wolf, and who can tell but that there
may be deadly danger lurking about her even now.
Christian’s smile grows brighter as it falls on Mary, “little Mary’s”
sparkling face, and her voice is happier and more musical in its modulation
as she answers her affectionate inquiries. They speak truly who say that
Christian has no thought of herself: at this hour Christian would fain be on
her knees in her solitary room, pleading for her lost brother; not lost, deaf
Christian, say not lost—is there not a lingering tone of sweet assurance in
thy mournful heart, which, if thou would’st but hear it, speaks to thee out of
the unknown secret stillness and says, Not lost, not lost, dear Christian,
though thou yet knowest not how the faithful One has answered thy
weeping prayers.
But, hush! little Mary is singing; a simple plaintive melody, as natural in
its pleasant notes, as the dropping of the withered leaves around her absent
brother, in yon far American forest. There is a charm in these old songs
which far surpasses more artistic music, for scarce is there a single ear on
which they fall that has not many remembrances and associations
awakened, or recalled, it may be joyful, it may be sorrowful, connected
with their simple measure and well-known words, and in such, and in no
other, does Mary Melville delight. There is one sitting by Mary’s side who
seems to comprehend what few of the listeners do, or care to do, the
singer’s delicate and sweet expression of the feeling of her well-chosen
song. He has never seen her before to-night, but he seems to have made
wonderfully good use of the short time he has spent beside her; and Mary
has already discovered that the gentleman-like stranger, who devoted
himself to her all through the evening, is a remarkably well-informed,
agreeable man, and quite superior to the frivolous youths who generally
buzz about in Elizabeth’s drawing-room, and form the majority of her
guests. He has brilliant conversational powers, this stranger, and the still
more remarkable art of drawing out the latent faculty in others, and Mary is
half-ashamed, as she sees herself led on to display her hoards of hidden
knowledge, adorned with her own clear perceptions of the true and
beautiful, which, unknown to herself, she has acquired. It is a strange, an
unusual thing with Mary to meet with any mind, save Christian’s, which
can at all appreciate her own, and she is rejoicing in her new companion’s
congenial temperament, and, in a little while, there is a group of listeners
collected round them, attracted by something more interesting than the
vapid conversations which are going on in this large room. Mr. Forsyth’s
accomplishments are universally acknowledged, and he shines resplendent
to night; and one after another, dazzled by his sparkling wit and still more
engaging seriousness, join the circle, of which Mary is still the centre.
“Who would have thought,” say we, with Mrs. James, as she gazes
wonderingly over the heads of her guests on the animated face of her young
sister-in-law,—“who would have thought that Mary knew so much, or could
show it so well!”
Is Christian’s care asleep to-night; what is she doing that she is not now
watching over her precious charge? No, it is not; her eyes, which have
strayed for a moment, are now resting fixed on Mary. See! how her cheek
flushes at that man’s graceful deference. Listen to the laugh that rings from
the merry circle at some sally of his polished wit. Mary looks grave and
anxious for a moment, for his jest has just touched something which she
will not laugh at, and he perceives it, and at once changes his tone, and
turns with polished ease the conversation into a new channel. Is it well that
Christian should be ignorant of one who is engrossing so much of her
sister’s attention? No, it is not; and she feels that it is not; so she calls
James, and is even now, while Mary’s joyousness is returning, anxiously
inquiring of her brother who this stranger is. James does not even know his
name. A cousin of Elizabeth’s brought him to-night, and introduced him as
a friend who had been of great service to him; then Elizabeth herself is
appealed to; Mrs. James is quite sure that Mr. Forsyth is a very respectable,
as well as a very agreeable man; he could never have found his way into her
drawing-room had he been other than that; her cousin never would have
brought him had he not been quite certain and satisfied on that point. He is
very rich, she believes, and very accomplished, she is sure, and, being
unmarried, she is extremely pleased to see him paying so much attention to
Mary. Christian shudders—why, she does not know; but she feels that this is
not well, there is a something in his look—such nonsense! But Christian has
always such strange, such peculiar notions, and is so jealous of all that
approach Mary.
The gay young people that are around Mary make room for Christian, as
she glides in to sit down by her sisters’s side. She is very grave now, as
always; but some of them have heard her story, and all the nature in their
hearts speaks for her in tones of sympathy, and their voices are quieter
always when beside her. Over most of them she has some other power
besides this of sympathetic feeling; there is hardly one there to whom she
has not done some deed of quiet kindness, which would not even bear
acknowledgment; thus they all love Christian. She sits down by Mary’s
side, and her heart grows calmer, and more assured again; for Mary bends
over her, and seeks forgiveness for her momentary forgetfulness. Pardon
from Christian is easily obtained; yet, gentle as she is, it seems not so easy
to win her favour. Mr. Forsyth’s fascinating powers, displayed and exerted
to the full, are all thrown away. See how coldly she listens to and answers
him; nay, how impatient she is of his courteous attentions. What has he
done wrong? what can ail Christian?
Mr. James Melville’s party has been a very brilliant one; but it is all over
now: the street grows suddenly sombre and silent opposite the darkened
windows, and Mrs. James is not in the sweetest of moods: the baby, now
that all the other music has ceased, is exercising his vigorous lungs for the
amusement of the tired household; his weary mamma is aggravated into
very ill-humour, and unfortunately can find no better way of relieving
herself, nor any better object, than by railing at Christian’s folly. Mrs. James
is sure, if Mr. Forsyth were to think of Mary Melville, they might all of
them be both proud and pleased, for he would be an excellent match for her.
She could not think what Christian expected for her—some unheard-of
prodigy she fancied, that nobody but herself ever dreamt of—thus did the
lady murmur on to the great annoyance of James.
But we must leave Mrs. James and her indignation to themselves, that
we may follow the sisters home. They had little conversation on the way.
Christian was silent and absorbed in her own thoughts, and Mary wondered,
but did not disturb her; for Mary, too, has thoughts unusual, which she cares
not to communicate; and soon, again, we are in the old room, no way
changed since we saw it first, three years ago; and Mr. Melville—how shall
we excuse ourselves for passing him over so lightly and so long—is here
unaltered, as much a fixture in his wide, soft chair, as any piece of furniture
in the well-filled room; and Robert, we lost him amid the belles of Mrs.
James’s party! but here he is again, distinct, full grown and manly, and still
retaining the blithe look of old. Christian alone has yet a disturbed
apprehensive expression on her usually calm and placid face, and she
wonders,
“How can James like such parties? it is so different from his wont.”
“Yes,” says Mary innocently, “I wonder that Elizabeth likes them. If
there were just two or three intelligent people like Mr. Forsyth, it would be
so much better.”
Poor Christian!
The protection of the Almighty has been implored “through the silent
watches of the night,” and Mr. Melville’s household is hushed in sleep—all
but Christian; for this quiet hour when all are at rest, is Christian’s usual
hour of thoughtful relaxation and enjoyment. But she had a clouded brow
and an uneasy look when she entered her room to-night—that room of
many memories. At length there is no mist of disquietude to be seen upon
her peaceful face; no doubt in her loving heart: she has gone to the footstool
of the Lord, and borne with her there that child of her tenderness and
affection, over whose dawning fate she has trembled, and has committed
her into the keeping of the Father of all; and she has poured forth, with
weeping earnestness, the longings of her soul for that lost brother, whom
even yet she knows not to be within the reach of prayer. Often has she
thought that Halbert may be dead, since day after day these years have
come and gone, and no tidings from, or of him, have gladdened her heart.
Her spirit has been sick with deferred hope, as month after month went by
and brought no message. But she is calmer to-night; the load is off her soul;
she has entrusted the guardianship of the twain into His hands who doeth all
things well, and with whom all things are possible; and wherefore should
she fear!
The light in her chamber is extinguished, and the moonbeams are
streaming in through the window. A few hours since she watched their
silvery radiance stealing, unheeded and unseen, into yon crowded room,
drowned in the flood of artificial light which filled it, and then she had
thought these rays an emblem of Heaven’s Viceroy—conscience—unknown
and unnoticed, perchance, by those gay people round about her, but even
then marking with silent finger upon its everlasting tablets, the hidden
things of that unseen and inner life in long detail, moment, and hour, and
day, for each one of them. But now, in the silence of her own room, these
beams have another similitude to Christian, as they pour in unconfined,
filling the quiet chamber. They tell her of peace, peace full, sweet, and
unmeasured,—not the peace of a rejoicing and triumphant spirit,—the
sunbeams are liker it,—but of one borne down with trial and sorrow, with a
sore fight of affliction, with a fear and anguish in times past, yet now at rest.
Oh, happy contradiction! distracted with cares and anxieties, yet calm amid
them all, full of the memories of bygone sorrow, of forebodings of sorrows
yet to come, but peaceful withal, how blessed the possession!
It falls upon her form, that gentle moonshine, and her features are lit up
as with a twilight ray of heaven: it lingers over her treasures as though it
loved them for her sake. It streams upon that portrait on the wall, and
illuminates its pensive and unchanging face, as with the shadow of a living
smile; and Christian’s heart grows calm and still within her beating breast,
like an infant’s, and holy scenes of old come up before her liquid eyes, like
ancient pictures, with that steadfast face upon the wall shining upon her in
every one; not so constant in its sad expression, but varying with every
varying scene, till the gathering tears hang on her cheeks like dewdrops,
and she may not look again.
And there is peace in that household this night, peace and sweet serenity,
and gentle hopefulness; for a blessing is on its prayer-hallowed roof and
humble threshold, and angels stand about its quiet doorway, guarding the
children of their King—the King of Kings.
CHRISTIAN MELVILLE.
EPOCH IV.
There is no emblem of our lives so fit

As the brief days of April, when we sit
Folding our arms in sorrow, our sad eyes
Dimmed with long weeping; lo! a wondrous ray,
Unhoped-for sunshine bursting from the skies
To chase the shadow of our gloom away.
And lest the dazzling gladness blind us, lo!
An hour of twilight quiet followeth slow,
Moistening our eyelids with its grateful tears,
Strengthening our vision for the radiant beam
That yet shall light these unknown future years,—
Each joy, each grief, in its appointed room,
Ripening the precious fruit for heaven’s high harvest home.
CHAPTER I.
Benedict. Pray thee, sweet Mistress Margaret, deserve well

at my hands by helping me to the speech of Beatrice.
* * * * *
Sweet Beatrice, wouldst thou come when I called thee?
Much ado about Nothing.
E are half inclined to lament that the incidents of our story

confine us to one short month, nay, oftener to one little day of
every passing year, but nevertheless so it is, and we may not
murmur. Doubtless could we have sketched the glories of some
midsummer morning or autumnal night, or wandered by our heroine’s side
through the gowan-spotted braes in the verdant springtime, we should have
had pleasanter objects to describe, and a pleasanter task in describing them,
and our readers a less wearisome one in following us; but seeing that we
must, perforce, abide by “the chamber and the dusky hearth,” even so, let it
be. The hearth of our present sketch is in nowise dusky, however; there is
nothing about it that is not bright as the blazing fire itself. If you look from
the window you may see that everything without is chained down hard and
fast in the iron fetters of the frost, and covered with a mantle of dazzling
whiteness. With tenacious grasp the wintry king fixes the less obdurate
snow to the heavy housetops, decking them as with hood and mantle; with
malicious glee it rivets each drop of spilt water on the slippery pavement,
bringing sudden humiliation, downfall and woe, to the heedless passengers;
and from the southern eaves where the sun has for some short time exerted
a feeble power, hang long icicles in curious spirals, like the curls of
youthful beauty. Keen and cold, it revels in the piercing wind, which
coming from the bleak north in full gush round the chill street corner,
aggravates the wintry red and blue which battle for the mastery in the faces
of the shivering passengers, and screams out its chill laughter in the gale,
when some sturdy man who has but now chased its little glowing votaries
from their icy play is suddenly overthrown himself by one incautious step,
and with prostration lower than Eastern does homage to its power, to the
great and loudly expressed satisfaction of the urchins aforesaid, who have
resumed again their merry game with renewed zeal and vigour.
It is just the kind of morning to make dwellers at home hug themselves
on their comfortable superiority over those whom necessity calls abroad, to
dare the dangerous passage of these treacherous streets and meet the rough
encounters of the biting wind. The room we stand in is the very picture of
neatness and comfort; a beautiful infant of two years old is roaming with
unsteady step about the bright fireside and over the carpet, a wide world to
him, intently making voyages of discovery hither and thither, among the
chairs and tables, the continents and islands of his navigation; and beside a
pretty work-table, with her delicate fingers employed in still more delicate
work, sits Mrs. James Melville, her brow furrowed and curved in
deliberative wisdom, giving earnest heed to schemes which are being
poured into her attentive ear, and ever and anon responding with oracular
gravity. Who is this that seeks and has obtained the infinite benefit of Mrs.
James’s counsel, and that now with deferential courtesy lays before her the
inexpressible advantages he will derive from her advice and assistance, and
insinuates the unending gratitude of which he has already given earnest in
delicate and well-timed presents, such as delight a lady’s heart? He is
speaking of a brilliant establishment to be offered to some one whom he
seeks to win, and shall win all the more easily through his kind friend Mrs.
James’s advice and co-operation. He is speaking of wealth which hitherto
he laments,—and here the petitioner sighs and looks, or tries to look
pathetic,—he has not properly employed, wherewith that as yet nameless
third party shall be endowed, and he winds up all with an eulogium upon
the extraordinary ability, and undeserved, but not unappreciated kindness of
the lady who smiles so graciously at his well-timed compliments. Mrs.
James is completely won over, and her full assistance and co-operation
pledged, for the pleader is skilled in his craft, and wont to be successful.
Who can resist Mr. Forsyth’s eloquence and special reasonings? The work
of consultation goes on, the toils are laid for Mary, sweet Mary Melville’s
unwitting feet, and Forsyth, on the strength of his ally’s assurances, has
already brightened in anticipatory triumph, and if all things be as Mrs.
James says they are, and all Forsyth’s promises be realised, is not Mary’s lot
a bright one? Nay, but is this a man to hold in his hands the happiness of
Christian’s sister?
Mrs. James is determined to signalise herself as a match-maker, and
there are a thousand captivating circumstances which conspire to make her
eager in the furtherance of Forsyth’s suit. She reckons up some of them:
First, it will really be an excellent settlement for Mary; where among her
father’s hum-drum acquaintance could she ever have found one anything at
all like so good; secondly, Mrs. Forsyth’s wealth and style will bring even
her, Mrs. James Melville, into a more brilliant sphere; and above all, there
will be the crowning delight of overcoming, or rather being able to set at
nought, all Christian’s opposition. Mrs. James, self-confident as she is, very
bold, and even impertinent as she can be at some times, and strong in the
might of superior elegance and beauty, has always been awed in the
presence of Christian’s quiet dignity, and this had annoyed and galled her
greatly. There is something in that grave dignity which she cannot
comprehend, and still more aggravating is the fact, that do what she will,
she cannot quarrel with her gentle sister-in-law, and that all her innuendoes
fall pointless and harmless. Christian will not hear Mrs. James’s petulance,
be it ever so loud, for with one calm word she shows her its insignificance;
she smiles at her sarcasms against old maids, as she might smile at some
nick-name of childish sport; nay, sometimes, and it is the nearest approach
to mirth which Christian is ever known to make now, she will turn round in
defence of the maligned sisterhood, and chase with lightfooted raillery,
which savours of days of old, the heavy wit of her opponent off the field.
Mrs. James never saw Christian ruffled or disturbed by any speech of hers,
save on that occasion which introduced Forsyth to Mary, and she was too
watchful and too much delighted to let the opportunity of prolonging her
annoyance cease; and Mary, a frequent visitor at her brother’s house, has
since that time, nearly a year now, met her sister-in-law’s accomplished
acquaintance so often, that people begin to whisper about Forsyth’s
devotion, and to look forward to a bridal; and when he is spoken of before
Mary, they smile and look in her face, and the colour on her soft cheek
deepens, and the blood flushes on her forehead, and then when they wonder
at his versatile talents, as they often do, for he is intellectually in that
society a giant among dwarfs, Mary’s downcast eyelids grow wet with
pleasant moisture, and her heart thrills with pleasure, so that she, loving
Christian as she does, is unconsciously furthering Mrs. James in her plan of
annoyance. Our poor Mary!
But we are neglecting the conversation which is still going on between
Mrs. James and her visitor. Forsyth is preparing to go, his visit has been
already prolonged beyond all usual bounds, yet he lingers still,
endeavouring with his persuasive eloquence to bring about one other
arrangement.
“You will bring Mary here to meet me, on new year’s day morning, my
dear madam?” he says softly, and in the most insinuating tone, “will you
not?”
“New year’s morning,” interrupted Mrs. James, “that will never do. You
know I have always a party on the new year’s night, I shall not be able to
give you that morning.”
“Well,” answered Forsyth, as smoothly and persuasively as he could,
“but if you could give us your presence for a few minutes, Mary and I, I
hope, will be able to manage the rest ourselves, and you know, my dear
Mrs. Melville,” he added still more blandly, “I am anxious to come to an
understanding with Mary as soon as possible. Come, you must add this to
the many kindnesses you have done me already. You will consent, I see.”
Mrs. James could not resist. “Well then, on new year’s morning be here,
and Mary shall meet you,” she said, and her gratified friend bows over her
extended hand. “You may come, Mr. Forsyth, on new year’s morning.”
Mr. Forsyth can never sufficiently express his obligation; and having
succeeded in all things according to his wish with Mrs. James Melville, he
takes his leave at last, and rejoices as he hurries through the streets, so cold
and bitter to other passengers, but so bright and cheerful to him in his
present mood, that soon now he will be assured of Mary. He has no doubt
about it, none at all, and he is certain that all that he wants is just this
opportunity which Mrs. James is to secure him, and then Mary Melville will
be his own, plighted and pledged his own.
It is but a few days, yet new year’s morning is as tardy in approach as if,
so big with fate to that young, ingenious, and unfearful spirit, it lingered on
its way willing to prolong her state of happy unconsciousness. The elegant
Mr. Forsyth yawns through the long weary days; though it is the time of his
own appointing he is impatient and restless, and his yawning and
irksomeness is redoubled on that dull, cold, cheerless evening before its
dawn, and he gets really nervous as the time draws near. Strange that one so
practised in the world, whose heart has been so long a very superfluous
piece of matter, should have his dead affections so powerfully awakened by
the simple grace and girlish beauty of guileless Mary Melville. Strange,
indeed, and if he is successful in winning her—as who can doubt he will—
what hope is there for our sweet Mary when his sudden vehement liking
passes into indifference. Poor Mary’s constant heart should be mated only
with one as warm and as full of affection and tenderness as itself; but who
shall have the choosing of their own future—alas, who! or who, if the
choice was given them, would determine aright?—not Mary. But there is a
power, the bridegroom in anticipation wots not of, ordering the very words
which shall fall from his lips to-morrow, overruling the craftiness of his
crafty and subtle spirit, and guarding the innocent simplicity of the prayer-
protected girl, defending her from all ill.
CHAPTER II.
York. I’ll not be by, the while; My Liege, farewell;

What will ensue hereof, there’s none can tell;
But by bad courses may be understood
That these events can never fall out good.
King Richard the Second.
EW YEAR’S day at last arrived, the time so anxiously waited for

by Forsyth; a cold clear winter morning; and Mary, invited
specially by her sister-in-law, leaves home to help—to help in
some little preparations for the evening, was the reason or plea
assigned by Mrs. James to secure Mary on that morning; and even Christian
had nothing to object to a request so reasonable, though it must be said that
Christian did not like her sister to be much among Mrs. James’s friends.
Nor had Mary herself been wont to like it either, but the Mary of a year ago
is not the Mary of to-day; she has not grown indifferent to Christian’s
wishes; very far from that, Mary was perhaps more nervously anxious to
please Christian than ever in all lesser things; she felt that a kind of
atonement, a satisfaction to her conscience, for her encouragement of the
one engrossing feeling of her heart, of which she dared not indeed seek
Christian’s approval. For the thought that in this most important particular
she was deceiving, or at least disingenuous to her dearest friend, concealing
from her what it so concerned her to know, gave Mary, acting thus contrary
to her nature, many a secret pang. But though this secret clouded her brow
and disturbed her peace at home, she hid it in her own heart. Still how
strange that Mary should be lightsome and happier with her brother’s wife,
whose character was in every respect so inferior to her own, than with her
gentle sister; yet so it was, and Mary’s heart beat quicker when she entered
James’s house, and quicker still when she saw there was some other visitor
before her. Who it was she needed not to ask, for Forsyth sprung to her side,
as she entered the cheerful room, with low-voiced salutation, and a glance
that brought the blush to her cheek, and caused her fair head to bend over
the merry little boy that came running to her knee, and hailed her as “Aunt
Mary.”
“Call me uncle, James, that’s a good little fellow, call me Uncle Walter,”
said Forsyth.
Mary’s blush grew deeper; but James the younger was said to resemble
Aunt Christian in many things, and in nothing more than in disliking
Forsyth; and he was not to be conciliated, either with sugar-plum or toy, but
remained steadfast in his childish instinct of dislike, so he said bluntly,
“No,”—a bad omen this; but Forsyth was not to be discouraged, and Mrs.
James, nettled a little by it, proceeded at once to open the campaign. Some
new music was lying on the table, and she pointed to it.
“See, Mary, here is a present from Mr. Forsyth,” she said, laughingly,
“but there is a condition attached to it which depends on you for its
fulfilment.”
Mary, glad of anything to hide her confusion, bent over the table to look
at it. “Well,” she said, “and what is the condition that depends on me.”
“Nay, ask the giver,” said Mrs. James, “he must make his agreement
with you himself, I cannot make bargains for him.”
Mary was half afraid to lift her eyes to Forsyth’s face, but she did so, and
asked by a glance what it was he required.
“The condition is not a very difficult one,” said he, in his most bland and
soothing tone, “it was merely that Mrs. Melville would get you to sing this
song for me. I was afraid I should fail did I ask myself.”
“And why this song, Mr. Forsyth,” asked Mary, “is it such a favourite?”
“I heard you sing it a year ago,” was the answer, spoken too low, Mary
thought, to reach Mrs. James’s ear, and again the blood came rushing in
torrents to her face.
Mrs. James began to move about as though about to leave the room; this
silence would not do, it was too embarrassing, and Mary resumed, though
her voice had likewise grown imperceptibly lower. “Christian is very fond
of this song, and we all of us like it because she does.”
Mrs. James heard this, however, and, elated by Mary’s coming to her
house that morning, and her own expected triumph over Christian, she
could not resist the temptation. “Oh, Christian has such strange notions,”
she said gaily, “she likes things that nobody else does. I can’t conceive why
you are all continually quoting Christian—Christian! one hears nothing else
from James and you, Mary, but Christian, Christian.”
“Christian never set her own inclination in opposition to any other
person’s wish in her life,” said Mary, warmly; “you do not know Christian,
Elizabeth, or you would not speak of her so.”
“Miss Melville’s good qualities,” chimed in Forsyth, “Miss Melville’s
rare qualities, must gain as much admiration wherever she is seen, as they
seem to have gotten love and reverence from all who are within the range of
their beneficent exercise, and who have the privilege of knowing their value
fully;” and he smiled his sweetest smile in Mary’s face, as she looked up to
him with grateful glistening eyes, and inwardly thanked him for his
appreciation of dear Christian in her heart.
How superior, thought Mary, is he to such worldly people as Elizabeth,
and her coterie, he appreciates Christian, he can estimate her properly. Yet
Mary, all the time that her heart glowed under these feelings towards
Forsyth, felt that she had thwarted Christian’s warmest wishes, and is still
farther thwarting them by the very look with which she thanked Forsyth for
his championship. Mrs. James is at the window carefully examining the
leaves of some rare winter plants—another gift of Forsyth’s giving; and
there ensues another awkward silence. At length she breaks in once more.
“Am I to have my music, Mary? will you fulfil the conditions Mr.
Forsyth has attached to this, or shall I have to send it back again?”
Forsyth is leaning over her chair, anxiously waiting for her answer. Mary
is at a loss what to do, but cannot say, No. Again Mrs. James is occupied
with the flowers.
“This is an era with me, Miss Melville,” Forsyth whispered in Mary’s
ear; “this day twelve months I first saw you.”
Mary’s fingers still hold the music, but the sheets tremble in her hands.
“Is it, indeed?” she says. “Oh, yes! I remember, it was at Elizabeth’s annual
party! It is an era to us all, also. We too have many recollections connected
with the New Year, but they are all sorrowful.”
“Not mine,” returned Forsyth. “Do you know, Miss Melville, I was much
struck then by your resemblance to a young man I once knew in Edinburgh,
a very fine gentleman-like lad of your own name too. I often wonder what
has become of him. I had some hand in inducing him to change some
ridiculously rigid opinions of his; when a fit of superstitious fear came over
him, and I believe his regard for me changed to a perfect hatred.”
Here Mr. Forsyth looked over to Mrs. James, as much as to say, it was
full time for her to go away.
The light is swimming in Mary’s eyes, everything before her has become
dim and indistinct; and she trembles, not as she trembled a moment since,
with agitated pleasure—it is horror, dread, fear that now shakes her slender
frame, and looks out from her dim and vacant eyes. There is no trace now
of the blush which wavered but a little ago so gracefully upon her cheek, it
is pale as death, as she sinks back into her chair. Forsyth and Elizabeth both
rushed to her side. What is, what can be, the matter?
“Nothing, nothing, I shall be better immediately,” she said, shuddering
as she raised herself up again, and drew away the hand which Forsyth had
taken; “I am better now, much better.”
A look of intelligence and mutual congratulation passed between her
companions. Poor thing, she is agitated, and out of sorts with the novelty of
her position; but what matters that, they are quite sure of Mary now, and
Mrs. James glides quietly out of the room.
As soon as she has gone, and they are left alone together, Forsyth with
all the eloquence of look and tone and gesture he can command, pours his
suit into Mary’s ear. How entirely will he not be devoted to her, to her
happiness. How perfectly does she reign in his affections; but it seems,
unless from a shiver, which thrills through her frame from time to time, that
he speaks to a statue, alike incapable of moving from that charmed place, or
of articulating anything in answer to his petition. Forsyth becomes alarmed,
and entreats, beseeches her to speak to him, to look at him only, to return
the pressure of his hand, if nothing more definite is to be said or done; and
suddenly Mary does look up, pale and troubled though her countenance be,
into his face, and speaks firmly:—
“Where, Mr. Forsyth,” she said, gazing at him as though she could
penetrate the veil, and read his inmost heart; “where did that young man go,
that you were speaking to me of just now; the one,” she added, with hasty
irritation, as she marked his astonished and deprecating gesture—“the one
you thought resembled me; to what place or country did he flee? Answer
me.”
“Mary, dear Mary!” pleaded Forsyth, “why ask me such a question now?
why terrify me with such looks. That superstitious fellow can be nothing to
you; and you, dear Mary, are all in all to me.”
Mary’s voice is still trembling, notwithstanding her firmness, and the
very force of her agitation has made it clear. “Where did he go to?” she
repeats once more.
“I do not know; I believe to America, the universal refuge,” answered
Forsyth, half angrily. “But why do you torment me thus, and answer my
entreaties by such questions? What has this to do with my suit? Will you
not listen to me, Mary?”
As he spoke, she rose with sudden dignity, and repelled the proud man
who subdued and supplicating half knelt before her. “Much, Sir,” she said,
with emphasis; “it has much to do with what you have said to me. I, to
whom you address your love—I, who have been deceived into esteeming
you so long—I, am the sister of Halbert Melville; of the man whom your
seductions destroyed!”
It is too much, this struggle, the natural feeling will not be restrained,
and Mary Melville hides her face in her hands, and tries to keep in the
burning tears. Forsyth has been standing stunned, as though a thunderbolt
had broken upon his head, but now he starts forward again. She is melting,
he thinks, and again he takes her hand in his own. It is forced out of his hold
almost fiercely, and Mary, again elevated in transitory strength, bids him
begone; she will not look upon the destroyer of her brother with a
favourable eye, nor listen to a word from his lips.
A moment after, the passengers in the street are turning round in
astonishment, to look at that face so livid with rage and disappointment
which speeds past them like a flash of lightning, and Mrs. James Melville
was called up to administer restoratives to her fainting sister—sweet gentle
Mary.
CHAPTER III.
If I may trust the flattering eye of sleep,

My dreams presage some joyful news at hand;
My bosom’s lord sits lightly in his throne;
And, all this day, an unaccustom’d spirit
Lifts me above the ground with cheerful thoughts.
Romeo and Juliet.
HRISTIAN MELVILLE is seated alone by her fireside, engaged

in her usual occupations, and full of her wonted thoughts; but her
present anxiety about Mary has taught her to linger less in the
past, and to look oftener forward to the future than she has been
accustomed to do heretofore, since sorrow made that once bright prospect a
blank to her. Nay, Christian, in her happier hours, has grown a dreamer of
dreams, and all her architectural fancies terminate in the one grand object,
the happiness of Mary. She sees the imminent danger she runs of having to
relinquish her one remaining treasure, and that into the keeping of one she
distrusts so much as Forsyth. Christian cannot tell how it is that she has
such an unaccountable, unconquerable aversion to him. True, his name is
the same as that of Halbert’s tempter; and association is the root, doubtless,
of all her prejudice—as prejudice everybody calls it—and Christian tries, as
she has tried a hundred times, to overcome her repugnance, and to recollect
the good traits of character that have been told her of him, and to school her
mind into willingness to receive him as Mary’s choice; and she breathes,
from the depths of her heart, the fervent petition for guidance and
deliverance so often repeated for her innocent Mary—her child, her sister—
and then her thoughts speed away, and Halbert rises up before her mental
vision. What can be his fate? Long and wearily does she ponder, and bitter
fancies often make her groan in spirit as one burdened. Is he still a living
man?—still to be hoped and prayed for; or, is Halbert now beyond all
human hope and intercession? Her heart grows sick and faint as she thinks
of the possibility of this; but she almost instantly rejects it; and again her
soul rises to her Lord in earnest ejaculations. Oh! but for this power of
prayer, but for this well-ascertained certainty, that there is One who hears
the prayers of his people, how should Christian Melville have lived
throughout these three long anxious years; how should she have endured the
unbroken monotony of every uneventful day, with such a load upon her
mind, and such fancies coming and going in her heart; how possibly
subdued the longings of her anxious love through all this time of waiting
and suspense? But her prayer has never ceased; like the smoke of the
ancient sacrifice, it has ascended continually through the distant heaven: the
voice of her supplications and intercedings have risen up without ceasing;
and surely the Hearer of prayer will not shut his ears to these.
There is some commotion going on below, the sound of which comes up
to Christian in a confused murmur, in which she can only distinguish old
Ailie’s voice. At first she takes no notice of it; then she begins to wonder
what it can be, so strange are such sounds in this quiet and methodical
house, though still she does not rise to inquire what it is. Christian is
engrossed too much with her own thoughts; and as the sounds grow more
indistinct, she bends her head again, and permits herself to be carried away
once more in the current of her musings. But the step of old Ailie is coming
up the stairs much more rapidly than that old footstep was wont to come;
and as Christian looks up again in astonishment, Ailie rushes into the room,
spins round it for a moment with uplifted hands, sobbing and laughing
mingled, in joyful confusion, and then dropping on the floor, breathless and
exhausted with her extraordinary pirouetting, throws her apron over her
head, and weeps and laughs, and utters broken ejaculations till Christian,
hastening across the room in great alarm to interrogate her, afraid that the
old woman’s brain is affected,
“What is the matter, Ailie?” Christian asks. “Tell me, what is the
matter?”
“Oh, Miss Christian!” and poor Ailie’s wail of sobbing mixed with
broken laughter sounded almost unearthly in Christian’s ear. “Oh, Miss
Christian! said I not, that the bairn of sae monie prayers suld not be lost at
last?”
“Ailie! Ailie! what do you mean? Have you heard anything of Halbert?”
and Christian trembled like a leaf, and could scarce speak her question for
emotion. “Ailie! I entreat you to speak to—to answer me.”
And Christian wrung her hands in an agony of hope and fear, unwitting
what to think or make of all this almost hysterical emotion of the old
faithful servant, or of her enigmatical words. “Look up, dear Christian; look
up!” Ailie needs not answer. Who is this that stands on the threshold of this
well-remembered room, with a flush of joy on his cheek, and a shade of
shame and fearfulness just tempering the glow of happiness in his eyes?
“Halbert!”
“Christian!”
The brother and sister so fearfully and so long separated, and during
these years unwitting of each other’s existence even, are thus restored to
each other once more.
A long story has Halbert to tell, when Christian has recovered from her
first dream of confused joy, a three year long story, beginning with that
fearful night, the source of all his sorrows and his sufferings. Christian’s
heart is bent down in silent shuddering horror as he tells her of how he fell;
how he was seduced, as by the craftiness of an Ahithophel, into doubt, into
scoffing, into avowed unbelief, and finally led by his seducer—who all the
previous time had seemed pure and spotless as an angel of light—into the
haunts of his profligate associates, so vicious, so degrading, that the blush
mantles on Halbert’s cheek at the bare remembrance of that one night. He
tells her how among them he was led to acknowledge the change which
Forsyth had wrought upon his opinions, and how he had been welcomed as
one delivered from the bondage of priestly dreams and delusions; how he
was taken with them when they left Forsyth’s house—the host himself the
prime leader and chief of all—and saw scenes of evil which he shuddered
still to think of; and how in the terrible revulsion of his feelings which
followed his first knowledge of the habits of these men, whose no-creed he
had adopted, and whose principles he had openly confessed the night
before, sudden and awful conviction laid hold upon him—conviction of the
nature of sin; of his sin in chief—and an apprehension of the hopelessness
of pardon being extended to him; and how, turning reckless in his despair,
he had resolved to flee to some place where he was unknown, uncaring
what became of himself. He told her then of his long agony, of his fearful
struggle with despair, which engrossed his soul, and how at last he was
prompted by an inward influence to the use of the means of grace once
more; and how, when at length he dared to open his Bible again, a text of
comfort and of hopefulness looked him in the face; that he had said to
himself, over and over again, “It is impossible!” till hope had died in his
heart: but here this true word contradicted at once the terrible utterance of
his self-abandonment. “All things,” it was written, “are possible with God;”
and Halbert told her, how the first tears that had moistened his eyes since
his great fall sprang up in them that very day. He told her of the scene so
fair, where this mighty utterance of the Almighty went to his soul, and
where he found peace; in the words of the gifted American—

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Centrifugal Separations in

Biotechnology - eBook PDF

Wallace Woon-Fong Leung

For Information on all Elsevier publications

Publisher: Susan Dennis

In God, I trust xvii

2 Principles of Centrifugal Sedimentation 27

3 Batch and Semibatch Centrifuges 49

4.2 Disk-Stack Centrifuge 75

5 Decanter Centrifuge 121

5.5 Differential Speed 124

6 Commercial Applications of Centrifugation in

7 Concentrating Solids by Centrifugation 161

8 Laboratory and Pilot Testing 173

9 Selection and Sizing of Centrifuges 203

10 Troubleshoot and Optimization 229

11 Visualization and Modeling of Flow and Separation in

11.5 Dimensionless Le Parameter 252

12 Disk-Stack Modeling 261

13 Performance Projection of Centrifuges in Bioseparation 273

13.4 Spintube 294

14 Rotating Membrane in Bioseparation 299

15 Flocculation With Decanter Centrifuges 331

15.3 Field Test 340

16 Case Studies of Monotonic and Unimodal Size

17 Classifying Bimodal Particle Size Distribution and Case

17.3 Processing Hybridoma Cell Broth 384

18 Integration of Unified Modeling With Practice in

18.3.5 Troubleshooting 429

Appendix A: Nomenclature 435

Since the Centrifugal Separation in Biotechnology (CSB) has been

flowable concentrate stream can be routed to a small diameter near the

parameters. These analytical forms include monotonic size distribution,

recombinant protein and other biotech processes, becomes available in

Wallace Woon-Fong Leung

In processing biological materials to produce high value-added

growing needs of centrifugation in combination with other separations and

was working on the manuscript among other responsibilities that also

Wallace Woon-Fong Leung

Centrifugal Separations in Biotechnology. DOI: https://doi.org/10.1016/B978-0-08-102634-2.00001-5

Figure 1.3 Escherichia coli cell schematic.

DNA technique. A schematic of E. coli bacteria cell is shown in

Extracellular proteins secreted from yeasts are produced for making

1.1.1 Common Host Cells for Secreting Recombinant Protein

Extracellular Intracellular Extracellular

Solid product Liquid product

Depth filter Centrifuge Centrifuge lysate Centrifuge

Drug substance and monoclonal antibodies

Figure 1.4A Drug substances produced from fermentation and down-

can be used for separation, clarification/polishing, thickening, classifica-

platforms with subsequent cellular expression of protein for labeling

1.1.2 Platform for Protein Expression

Extracellular Intracellular Intracellular

Figure 1.4B Three types of secreted recombinant protein by cells and

1.1.3 Extracellular Protein

1.1.4 Intracellular Protein—Liquid

For example Fine filtration

Figure 1.4C Extracellular protein secreted into broth.

high speed is necessary to generate large centrifugal force to carry out

1.1.5 Intracellular Protein—Inclusion Body

Figure 1.4D Intracellular protein secreted inside the cells.

subsequent processing to restore its bioactivity before use. At harvest, the

Figure 1.4E Intracellular protein secreted into the inclusion bodies.